WO2023128594A1 - Polypeptide for delivering antigen to immune cells - Google Patents
Polypeptide for delivering antigen to immune cells Download PDFInfo
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- WO2023128594A1 WO2023128594A1 PCT/KR2022/021480 KR2022021480W WO2023128594A1 WO 2023128594 A1 WO2023128594 A1 WO 2023128594A1 KR 2022021480 W KR2022021480 W KR 2022021480W WO 2023128594 A1 WO2023128594 A1 WO 2023128594A1
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
Definitions
- the present invention relates to polypeptides for delivering antigens to immune cells, and specifically, novel polypeptides including cell membrane penetrating peptides and peptides that bind to surface molecules of immune cells, and fusion polypeptides to which antigens are bound to the polypeptides.
- novel polypeptides including cell membrane penetrating peptides and peptides that bind to surface molecules of immune cells, and fusion polypeptides to which antigens are bound to the polypeptides.
- MTS membrane transduction sequence
- CPP cytoplasmic penetration peptide
- FGF Fibroblast Growth Factor
- MTS or CPP can be used as an antigen carrier.
- Peptide antigens introduced into the cytoplasm are processed through the proteasome and presented on the cell surface on MHC class I.
- it can be used for therapeutic substance delivery to deliver proteins with specific functions developed targeting various signal transduction processes that control cell death and cell growth in the cytoplasm.
- CTP cytoplasmic transduction peptide
- Staphylococcus aureus is widely distributed in the natural world because of its strong resistance to the natural environment. are very likely to be contaminated.
- the pathogenic factors produced by Staphylococcus aureus include exotoxin, enterotoxin, hemolysin toxin, leucodicin, and epidermolytic toxin, as well as toxic shock.
- TSST toxin shock syndrome toxin
- Staphylococcal enterotoxin B type (Staphylococcal Enterotoxin B or hereinafter referred to as 'SEB') is a toxin produced by a toxin-type food poisoning bacterium of Staphylococcus aureus , which is distributed worldwide and mainly causes food poisoning and abnormal immune response. can lead to serious autoimmune diseases.
- SEB is a protein of about 26 to 29 kDa in size and has a small molecular weight, but SEB is highly soluble in water, has strong resistance to protease enzymes such as pepsin, trypsin, and papain, and is resistant to extreme temperatures. It is a very stable toxin that tolerates pH well.
- SEB is named superantigen (SAg), and is associated with the T-cell receptor (TCR) present on activated CD4+ or CD8+ T cells and Antigen Presenting Cell (APC). It is an antigen that plays a role in linking between MHC class II (Major Histocompatibility Complex class II) present in ). SEB binds to MHC class II of macrophages and then binds to the receptor Vb8 of T cells, thereby abnormally activating T cells and producing IL-6, IL-1 ⁇ , MIP-1 (Macrophage Inflammatory Protein-1), etc. By secreting a large amount of the same amount of inflammatory cytokines, an abnormal immune response is induced. When the respiratory system is exposed to SEB, there are few people who die of SEB, but more than 80% of exposed people show clinical symptoms, and it is known that they show symptoms that are so severe that daily life is impossible for 1 to 2 weeks.
- the inventors of the present application have selected peptides that bind to MHC class II through structural studies of cell membrane penetrating peptides and SEB, and a novel polytype including cell membrane penetrating peptides and peptides that bind to surface molecules of immune cells It was confirmed that the peptide can improve the yield of antigen delivery to immune cells, and the present invention was completed.
- An object of the present invention is to provide novel polypeptides for delivering antigens to immune cells.
- An object of the present invention is to provide a nucleic acid encoding the polypeptide.
- An object of the present invention is to provide a fusion polypeptide in which an antigen is bound to the polypeptide.
- An object of the present invention is to provide a nucleic acid encoding the fusion polypeptide.
- An object of the present invention is to provide an immune cell containing the fusion polypeptide or the nucleic acid.
- An object of the present invention is to provide an immunotherapeutic agent comprising the immune cells.
- An object of the present invention is to provide an anti-tumor or anti-cancer vaccine comprising the immune cells.
- An object of the present invention is to provide a composition for treating tumor or cancer containing the immune cells.
- An object of the present invention is to provide a method for treating tumor or cancer comprising administering the immune cells to a subject.
- An object of the present invention is to provide a use of the immune cells for preparing a composition for treating tumor or cancer.
- the present invention provides a polypeptide for delivering an antigen to immune cells, including a cytoplasmic transduction peptide (CTP) and a peptide that binds to surface molecules of immune cells.
- CTP cytoplasmic transduction peptide
- the present invention provides nucleic acids encoding the above polypeptides.
- the present invention provides a fusion polypeptide in which an antigen is bound to the polypeptide.
- the present invention provides nucleic acids encoding the fusion polypeptides.
- the present invention provides an immune cell containing the fusion polypeptide or a nucleic acid encoding the fusion polypeptide.
- the present invention provides an immunotherapeutic agent comprising the above immune cells.
- the present invention provides an anti-tumor or anti-cancer vaccine comprising the above immune cells.
- the present invention provides a composition for treating tumor or cancer comprising the immune cells.
- the present invention provides a method for treating tumor or cancer comprising administering the immune cells to a subject.
- the present invention provides a use of the immune cells for preparing a composition for treating tumor or cancer.
- Figure 1 shows the results of comparing the MoDC cell membrane permeability of the CTP / CTP-SEB group.
- Figure 2 shows the difference in MoDC permeability of the CTP/CTP-SEB group.
- FIG. 3 shows a vector for producing a recombinant fusion protein.
- Figure 4 shows the results of purification and QC analysis using Ni-NTA resin in the production of recombinant fusion proteins.
- Figure 7 shows the results of analysis of the remaining amount of protein in the culture medium.
- Figure 8 shows the results of DC phenotype and cell membrane permeability comparison experiment of PTD series peptides.
- Figure 10 shows the results of confirming the antitumor effect in mice inoculated with dendritic cells sensitized with CTP-OVA or SCTP-OVA antigen.
- FIG. 11 shows the results of permeability analysis according to immune cell types.
- the present invention relates to a polypeptide for delivering an antigen to immune cells, including a cytoplasmic transduction peptide (CTP) and a peptide that binds to surface molecules of immune cells.
- CTP cytoplasmic transduction peptide
- the cell membrane penetrating peptide is, for example, a protein transduction domain, for example, CTP (cytoplasmic transduction peptide, cytoplasmic residual cell membrane penetrating peptide), HP4, Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia ), Pep-1 (peptide), PTD-5, R9 (arginine), and may be one or more selected from the group consisting of peptides containing the 7R domain, but is not limited thereto.
- CTP cytoplasmic transduction peptide, cytoplasmic residual cell membrane penetrating peptide
- HP4 cytoplasmic transduction peptide, cytoplasmic residual cell membrane penetrating peptide
- Hph-1 cytoplasmic residual cell membrane penetrating peptide
- Mph-1 Mph-1
- Sim-2 Tat
- Tat VP22
- Antp Antp
- Pep-1 peptide
- PTD-5 arginine
- the cell membrane penetrating peptide has cell membrane penetrating ability, shows cell membrane penetrating phenomenon even when a cell is treated with a proteolytic enzyme after a certain period of time, and remains in the cytoplasm after penetrating the cell membrane It may be a cytoplasmic residual cell membrane penetrating peptide having the characteristics of When the cell membrane penetrating peptide is fused with a peptide that binds to a surface molecule of an immune cell, endocytosis is inhibited and ubiquitin is inhibited at the same time. It was confirmed that the polypeptide is not degraded and exists in the cell .
- the cell membrane penetrating peptide may have a length of, for example, 9-20 aa, more preferably 9-15 aa, and most preferably about 11 aa.
- the cell membrane penetrating peptide may include, for example, a sequence selected from the group consisting of SEQ ID NOs: 5 to 7.
- the inventors of the present application found that when a cell membrane penetrating peptide containing a sequence selected from the group consisting of SEQ ID NOs: 5 to 7 is fused with a peptide that binds to a surface molecule of an immune cell, it exhibits superior cell membrane penetrating ability compared to the cell membrane penetrating peptide alone. confirmed that it can.
- the peptide that binds to the surface molecule of the immune cell may be, for example, a peptide that binds to a major histocompatibility complex (MHC) molecule. Specifically, it may include a peptide that binds to major histocompatibility complex (MHC) class II expressed as surface molecules of the immune cells.
- MHC major histocompatibility complex
- the peptide binding to the surface molecule of the immune cell may include a sequence selected from the group consisting of SEQ ID NOs: 1 to 4.
- the antigen may be a cancer or tumor antigen for activating an immune response against cancer or tumor.
- the antigen is a cancer or tumor antigen and can induce cancer or tumor specific immune cells, such as cytotoxic T cells, in cell therapy.
- the cancer or tumor antigen may be, for example, a self or non-self derived antigen.
- autologous cancer or tumor antigens derived from a patient's own gene include hTERT (GenBank: BAC11010.1), WT1 (GenBank: AAO61088.1), NY-ESO1 (GenBank: CAA05908.1), MAGE-A3 (NCBI Reference Sequence: NP_005353.1) and cancer-specific mutated antigens, such as neoantigens, mutated P53, and RAS, may include tumor suppressor or inducing genes, and cancer or tumor-inducing virus antigens such as foreign cancer or tumor antigens CMV, EBV, HPV, and the like.
- hTERT an autologous cancer or tumor antigen
- hTERT is an enzyme that synthesizes telomeric DNA at the ends of chromosomes, and cancer cells activate this enzyme excessively to avoid telomere-dependent apoptosis. It is known as a target antigen for various solid cancers (Kim NW, et al. Science. 1994;266:2011-2015), and WT1 is a gene related to Wilms tumor that encodes a zinc finger transcription factor, which causes cell proliferation and differentiation, apoptosis, and organs. As a protein involved in the development of cerebrospinal cancer, it is known as a target antigen for lung cancer, etc. (Call KM, et al., Cell. 1990.
- NY-ESO1 is one of the proteins belonging to CTA (cancer testis antigen) and is well known to be expressed in various cancer cells, including germ cells, sarcoma, and breast cancer (Gnjatic S, et al., Adv. Cancer Res. 2006;95:1-30).
- MAGE-A3 is a protein belonging to the melanoma-associated antigen family and is known to be overexpressed in various cancer cells, including lung cancer, sarcoma, and melanoma, and is being evaluated as a suitable target antigen for cancer immunotherapy (Decoster L, et al., Ann Oncol. 2012 Jun;23(6):1387-93).
- the antigen is, for example, A33, ALK, alpha-fetoprotein (AFP), adrenergic receptor beta 3 (ADRB3), alpha-folate receptor, AD034, AKT1, BCMA, beta-human chorionic gonadotropin, B7H3 (CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD79a, CD79b, CD89, CD97, CD123, CD138, CD160, CD171 , CD179a, carbonic anhydrase IX (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR4, C-type lectin-like molecule-1 (CLL-1 or CLECL1), claudin 6 (CLDN6), CXORF61 , CAGE, CDX2, CLP, CT-7, CT8/HOM-TES-85, cTAGE-1
- the antigen is a pathogen (bacterium, virus, etc.), a chemical substance, pollen, cancer cell, shrimp, etc., or some peptide or protein thereof, more preferably a cancer antigen peptide, or a substance that can cause an immune response in the body Not limited to this.
- a pathogen bacteria, virus, etc.
- a chemical substance, pollen, cancer cell, shrimp, etc. or some peptide or protein thereof, more preferably a cancer antigen peptide, or a substance that can cause an immune response in the body Not limited to this.
- the antigen may preferably be a protein, recombinant protein, glycoprotein, gene, peptide, polysaccharide, lipopolysaccharide, polynucleotide, cell, cell lysate, bacterium, virus, etc., more preferably, a cancer antigen peptide can be
- the protein includes antibodies, antibody fragments, structural proteins, regulatory proteins, transcription factors, toxin proteins, hormones, hormone analogues, enzymes, enzyme fragments, transport proteins, receptors, fragments of receptors, biological defense inducers, It may be a storage protein, a movement protein, an exploitive protein, a reporter protein, and the like. However, any material capable of inducing an immune response by acting as an antigen in a living body is not limited thereto.
- the antigen according to the present invention is an antigen capable of binding to the polypeptide according to the present invention, and may include inactivated tumor cells, tumor cell-related genes, peptides or proteins produced by genetic recombination.
- the nucleotide sequence encoding the antigen may be known, and the full length of the known sequence may be used, but a part of the full length may also be used.
- a nucleotide sequence encoding the antigen may be cloned into a vector to express the desired antigen.
- the antigen may bind to the N-terminus or C-terminus of the polypeptide.
- the covalent bonding method may be performed using a method known in the art depending on the type of antigen, and may be obtained, for example, by cloning and intracellularly expressing a gene encoding a polypeptide.
- a linker that does not interfere with the activity of the polypeptide for example N-succinimidyl iodoacetate, N-maleimidobutyryl oxysuccinamide ester, 1,5-difluoro-2,4 -Dinitrobenzene, disdiazobenzidine, 3,3-dithio-bis-(sulfosuccinimidyl-propionate), ethylene glycol bis(sulfosuccinimidylsuccinate), dicyclohexyl carbodiimide etc.
- N-succinimidyl iodoacetate N-maleimidobutyryl oxysuccinamide ester
- 1,5-difluoro-2,4 -Dinitrobenzene 1,5-difluoro-2,4 -Dinitrobenzene
- disdiazobenzidine 3,3-dithio-bis-(sulfosuccinimidyl-propionate
- a linker that can be cleaved in vivo is used.
- binders with carboxylic acid esters and/or disulfide bonds may be used.
- the immune cells may be antigen-presenting cells (APCs) that present antigens delivered by the polypeptide according to the present invention on their surface.
- APCs antigen-presenting cells
- the immune cells may be dendritic cells, monocytes, Wrangel Hans cells, macrophages, T cells, or B cells, but are not limited thereto.
- a peptide that binds to the surface molecule of the immune cell may be fused to one end of a cell membrane penetrating peptide, for example.
- the peptide that binds to the surface molecule of the immune cell may be bound to the N-terminus or C-terminus of the cell membrane penetrating peptide.
- the peptide binding to the surface molecule of the immune cell may be connected to the cell membrane penetrating peptide through a linker.
- the linker may be a peptide linker and may have a length of about 5-25 aa, specifically about 5-10 aa.
- hydrophilic amino acids such as glycine and/or serine may be included, but are not limited thereto.
- the linker is, for example, glycine linker (G, Gly) p (p is 1 to 10), GS linker (G n S) m (n, m is 1 to 10, respectively) to impart structural flexibility.
- the linker may include GGGGS or (GGGGS) 2 , or 5-10 aa of glycine where p is 5-10 in (G, Gly)p.
- the present invention relates to a nucleic acid encoding the polypeptide.
- Nucleic acid has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include not only natural nucleotides, but also analogs in which sugar or base sites are modified. .
- the sequence of a nucleic acid according to the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
- the nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or further expression. Based on this, the present invention relates to a vector comprising the nucleic acid in another aspect.
- Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
- the term "vector” refers to a plasmid vector as a means for expressing a gene of interest in a host cell; cosmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors.
- the nucleic acid encoding the polypeptide is operably linked to a promoter.
- “Operably linked” means a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence is linked to the other nucleic acid. It will regulate the transcription and/or translation of the sequence.
- a nucleic acid expression control sequence eg, a promoter, signal sequence, or array of transcriptional regulator binding sites
- a strong promoter capable of promoting transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
- a ribosome binding site for initiation of translation e.g., amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
- a promoter derived from the genome of a mammalian cell eg, metallotionine promoter, ⁇ -actin promoter, human hemoglobin promoter, and human muscle creatine promoter
- mammalian Promoters derived from animal viruses such as adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter from HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter from HIV , the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Rouss sarcoma virus (RSV) can be used, and usually has a polyadenylation sequence as a transcription termination sequence.
- a mammalian Cell eg, metallotionine promoter, ⁇ -actin promoter, human hemoglobin promoter, and human muscle creatine promoter
- mammalian Promoters derived from animal viruses such as
- vectors may be fused with other sequences to facilitate purification of the polypeptides expressed therefrom.
- Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).
- the vector contains an antibiotic resistance gene commonly used in the art as a selectable marker, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.
- the present invention relates to a cell transformed with the above-mentioned vector.
- Cells used to produce the polypeptides of the present invention may be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells.
- Escherichia coli strains of the genus Bacillus such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (eg, Staphylocus carnosus) may be used, but are not limited thereto.
- animal cells include COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S, or HT1080, but is not limited thereto no.
- the present invention relates to a fusion polypeptide in which an antigen is linked to the polypeptide.
- the antigen may be a cancer or tumor antigen for activating an immune response against cancer or tumor.
- the antigen is a cancer or tumor antigen and can induce cancer or tumor specific immune cells, such as cytotoxic T cells, in cell therapy.
- the cancer or tumor antigen may be, for example, a self or non-self derived antigen.
- autologous cancer or tumor antigens derived from a patient's own gene include hTERT (GenBank: BAC11010.1), WT1 (GenBank: AAO61088.1), NY-ESO1 (GenBank: CAA05908.1), MAGE-A3 (NCBI Reference Sequence: NP_005353.1) and cancer-specific mutated antigens, such as neoantigens, mutated P53, and RAS, may include tumor suppressor or inducing genes, and cancer or tumor-inducing virus antigens such as foreign cancer or tumor antigens CMV, EBV, HPV, and the like.
- hTERT an autologous cancer or tumor antigen
- hTERT is an enzyme that synthesizes telomeric DNA at the ends of chromosomes, and cancer cells activate this enzyme excessively to avoid telomere-dependent apoptosis. It is known as a target antigen for various solid cancers (Kim NW, et al. Science. 1994;266:2011-2015), and WT1 is a gene related to Wilms tumor that encodes a zinc finger transcription factor, which causes cell proliferation and differentiation, apoptosis, and organs. As a protein involved in the development of cerebrospinal cancer, it is known as a target antigen for lung cancer, etc. (Call KM, et al., Cell. 1990.
- NY-ESO1 is one of the proteins belonging to CTA (cancer testis antigen) and is well known to be expressed in various cancer cells, including germ cells, sarcoma, and breast cancer (Gnjatic S, et al., Adv. Cancer Res. 2006;95:1-30).
- MAGE-A3 is a protein belonging to the melanoma-associated antigen family and is known to be overexpressed in various cancer cells, including lung cancer, sarcoma, and melanoma, and is being evaluated as a suitable target antigen for cancer immunotherapy (Decoster L, et al., Ann Oncol. 2012 Jun;23(6):1387-93).
- the antigen is, for example, A33, ALK, alpha-fetoprotein (AFP), adrenergic receptor beta 3 (ADRB3), alpha-folate receptor, AD034, AKT1, BCMA, beta-human chorionic gonadotropin, B7H3 (CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD79a, CD79b, CD89, CD97, CD123, CD138, CD160, CD171 , CD179a, carbonic anhydrase IX (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR4, C-type lectin-like molecule-1 (CLL-1 or CLECL1), claudin 6 (CLDN6), CXORF61 , CAGE, CDX2, CLP, CT-7, CT8/HOM-TES-85, cTAGE-1
- the antigen is a pathogen (bacterium, virus, etc.), a chemical substance, pollen, cancer cell, shrimp, etc., or some peptide or protein thereof, more preferably a cancer antigen peptide, or a substance that can cause an immune response in the body Not limited to this.
- a pathogen bacteria, virus, etc.
- a chemical substance, pollen, cancer cell, shrimp, etc. or some peptide or protein thereof, more preferably a cancer antigen peptide, or a substance that can cause an immune response in the body Not limited to this.
- the antigen may preferably be a protein, recombinant protein, glycoprotein, gene, peptide, polysaccharide, lipopolysaccharide, polynucleotide, cell, cell lysate, bacterium, virus, etc., more preferably, a cancer antigen peptide can be
- the protein includes antibodies, antibody fragments, structural proteins, regulatory proteins, transcription factors, toxin proteins, hormones, hormone analogues, enzymes, enzyme fragments, transport proteins, receptors, fragments of receptors, biological defense inducers, It may be a storage protein, a movement protein, an exploitive protein, a reporter protein, and the like. However, any material capable of inducing an immune response by acting as an antigen in a living body is not limited thereto.
- the polypeptide and the antigen are linked through a covalent or non-covalent bond, form a complex, and are included in the immune cell, thereby sensitizing the immune cell with the antigen.
- a fusion polypeptide in which an antigen is bound to the polypeptide may be delivered through a vector.
- the present invention also relates to an immune cell comprising said fusion polypeptide and said nucleic acid.
- Immune cells can be sensitized through the antigen of the fusion polypeptide.
- the sensitization means that the antigen is delivered to immune cells and loaded onto the cell surface.
- the antigen may be prepared in the form of a polypeptide and a recombinant antigen, and sensitize immune cells.
- Immune cells can be sensitized by treating immune cells with the polypeptide and the antigen bound to the polypeptide for 24 hours or less. The sensitization time may be adjusted according to the type of antigen and the type of immune cell.
- the antigen is an antigen capable of binding to the polypeptide according to the present invention, and may include inactivated tumor cells, tumor cell-related genes, peptides or proteins produced by genetic recombination methods.
- the nucleotide sequence encoding the antigen may be known, and the full length of the known sequence may be used, but a part of the full length may also be used.
- a nucleotide sequence encoding the antigen may be cloned into a vector to express the desired antigen.
- the antigen may bind to the N-terminus or C-terminus of the polypeptide.
- the covalent bonding method may be performed using a method known in the art depending on the type of antigen, and may be obtained, for example, by cloning and intracellularly expressing a gene encoding a polypeptide.
- a linker that does not interfere with the activity of the polypeptide for example N-succinimidyl iodoacetate, N-maleimidobutyryl oxysuccinamide ester, 1,5-difluoro-2,4 -Dinitrobenzene, disdiazobenzidine, 3,3-dithio-bis-(sulfosuccinimidyl-propionate), ethylene glycol bis(sulfosuccinimidylsuccinate), dicyclohexyl carbodiimide etc.
- N-succinimidyl iodoacetate N-maleimidobutyryl oxysuccinamide ester
- 1,5-difluoro-2,4 -Dinitrobenzene 1,5-difluoro-2,4 -Dinitrobenzene
- disdiazobenzidine 3,3-dithio-bis-(sulfosuccinimidyl-propionate
- a linker that can be cleaved in vivo is used.
- binders with carboxylic acid esters and/or disulfide bonds may be used.
- the immune cells may be antigen-presenting cells (APCs) that present antigens delivered by the polypeptide according to the present invention on their surface.
- APCs antigen-presenting cells
- the immune cells may be dendritic cells, monocytes, Wrangel Hans cells, macrophages, T cells, or B cells, but are not limited thereto.
- the present invention relates to an immunotherapeutic agent comprising the immune cells.
- An immunotherapeutic agent according to the present invention may increase an immune response or selectively increase a portion of an immune response desirable for treatment or prevention of a particular disease, infection or disorder.
- the present invention relates to an anti-tumor or anti-cancer vaccine comprising the immune cells. Since the immunogenicity of a subject can be increased through the vaccine according to the present invention, proliferation and/or metastasis of a tumor in the subject can be prevented or suppressed.
- the vaccine may include both an immunization method performed by one-time administration and an immunization method performed by continuous administration.
- the present invention relates to a composition for treating tumors or cancer.
- the amount and administration method of the active ingredient included in the composition of the present invention can be determined by a person skilled in the art based on the symptoms and severity of the disease of a typical patient. It may also be presented in unit-dose or multi-dose containers, such as sealed ampoules and bottles.
- composition of the present invention can be administered orally or parenterally.
- the administration route of the composition according to the present invention is not limited to these, but for example, bronchial, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal , sublingual or topical administration is possible.
- the dosage of the composition according to the present invention varies in its range depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate or severity of disease, etc. can decide
- the composition of the present invention can be formulated into a suitable dosage form for clinical administration using known techniques.
- composition of the present invention is a pharmaceutical composition and may include a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier As those commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto.
- the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
- a suitable dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, administration time and administration route, but severe toxicity (Grade 3 or more) has not been reported, so much is determined by the manufacturing method and yield.
- the intradermal or subcutaneous dosage of the pharmaceutical composition of the present invention is preferably 0.1X107 to 10X107 cells per dose.
- the pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using pharmaceutically acceptable excipients according to a method that can be easily performed by those skilled in the art.
- the formulation may be in the form of suspension in a cell freezing solution or suspension in a buffer solution, and may additionally include a stabilizer.
- Immune cells according to an embodiment of the present invention may be frozen after antigen sensitization and thawed if necessary.
- the dosage of the active ingredient may be appropriately selected according to various factors such as the route of administration, age, sex, weight and severity of the patient, and the composition is known to have an effect of preventing, improving or treating tumors or cancer symptoms. It can be administered in parallel with the compound of
- a 96-well plate was coated with MHC class ⁇ protein, and then the peptide was treated with 50 nM and incubated for 24 hours. Then, after 3 times PBS washing, HRP-antibody (1:500) was incubated for 2 hours, after 5 times PBS washing, TMB solution was added, and after 10 ⁇ 30min incubation, color development using a stopping solution (2M H 2 SO 4 ) After stopping, the absorbance was confirmed at 450 nm to select peptides with good binding ability. As a result, among the five synthesized peptides, the peptide sequence corresponding to SEB2 showed the highest binding ability, followed by SEB4, SEB1, and SEB3 in order.
- monocytes were adhered to a T150 plate for 30 minutes using 1% AB RPMI medium. Thereafter, non-adherent cells were removed and cultured for 3 days in the maturation medium hGM-CSF 30ng/ml and hIL-4 20ng/ml.
- MoDC cells were seeded on coverslips in a 30-40% confluent state and stored in 6-well plates at a cell density of 1 x 10 4 cells/well.
- the peptides were treated and incubated according to each concentration, washed with PBS, and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Thereafter, after blocking using 3% BSA, staining was performed using FITC and DAPI to display peptides penetrated into cells, and then analysis was performed using a fluorescence microscope.
- MoDC was obtained by culturing dendritic cells after isolating monocytes from human peripheral blood. Using differentiated dendritic cells, each group was treated according to the peptide concentration and cultured for 10 minutes, and then each group was washed with PBS. To analyze the phenotype of dendritic cells, anti-mouse CD80, anti-mouse CD86, anti-mouse CD11c, and peptide-FITC were stained, respectively, and then measured using FACS Canto II. First, after confirming the phenotype of the prepared MoDC, an analysis was performed to confirm that the prepared DC functioned as a DC, and as a result, it was confirmed that all DCs showed a phenotype of 90% or more.
- a recombinant protein vector was commissioned (genscript, Piscataway, NJ) to produce a recombinant protein vector as shown in FIG.
- the recombinant gene was cultured using E.coli, produced in an IB state, and then separated and purified using Ni-NTA resin through a solubilization step. Specifically, after culturing the seed at 37 ° C overnight, 37 grown at °C. When the absorbance at OD600 nm reached about 0.8 to 1.0, protein expression was induced by adding IPTG to a final concentration of 0.5 mM, followed by incubation with shaking at 180 rpm for 8 hours.
- Bacterial cells grown to a value of 1.5 to 2.0 at an absorbance of OD600 nm were precipitated by centrifugation at 10,000 rpm and 4 ° C for 20 minutes, and then 50 ml of disintegration buffer (300 mM NaCl, 50 mM Tris) per 2 g (1 L culture volume) of the precipitated cells. -HCl (pH 8.0), 0.5% Triton X-100) and the cells were agitated until completely suspended. After adding PMSF to a final concentration of 1 mM, the cells were lysed with an ultrasonicator.
- disintegration buffer 300 mM NaCl, 50 mM Tris
- -HCl pH 8.0
- Triton X-100 0.5% Triton X-100
- the lysed cells were centrifuged at 12000 rpm, 4°C for 30 minutes to separate the suspension and the total lysate, and then collected only the suspension and filtered through a cellulose filtration membrane with a pore size of 0.45 ⁇ m, and equilibrated with an equilibration buffer beforehand. Nickel affinity chromatography loaded onto the column. After the purified protein was stabilized through buffer exchange, QC was performed. The results are shown in FIG. 4 .
- MoDC cells were seeded onto coverslips at 30-40% confluent and stored in 6 well plates at a cell density of 1 x 104 cells/well.
- the peptides were treated and incubated according to each concentration, washed with PBS, and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Subsequently, after blocking using 3% BSA, staining was performed using Cy5.5 and DAPI to display proteins permeated into cells, and then analysis was performed using a fluorescence microscope.
- the membrane was washed 4 times with TBST, treated with an optimal concentration of primary antibody diluted in 4% non-fat dry milk, and incubated overnight at 4°C. After washing four times with TBST, the cells were incubated with HRP-conjugated secondary antibodies for 45 minutes. Bound antibodies were detected using chemiluminescent HRP substrate (Millipore, USA) and Chemi DOC (Bio-RAD, USA).
- protease inhibitor cocktail [2 mM AEBSF, 1 mM EDTA, 130 ⁇ M Bestatin, 1 ⁇ M leupeptin, 14 ⁇ M E-64, 0.3 ⁇ M Aprotinin] and NP-40 lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40) containing dephosphatase inhibitors (Roche). 40, 10% glycerol), the cells were put on a rotator in a 4 ° C chamber and rotated for 30 minutes (C1, 25 rpm).
- Protein A/G beads (Pierce) was added and returned to the rotor in a chamber at 4° C. for 1 to 2 hours.
- NP-40 lysis buffer solution was washed three times at 1200 rpm, 3 min, and 4 ° C. After washing, the remaining buffer was completely removed, and 20 ⁇ l of 5X sample buffer was added and boiled. After cooling on ice and centrifuging for 2 minutes, only the supernatant was collected and separated by 7.5-12% SDSPAGE method. The gel was again blotted with a PVDF membrane, and after attaching a primary antibody to the protein-transferred membrane, an antibody to the secondary antibody IgG was attached, and then observed with an ECL detection kit.
- Dendritic cells were cultured by differentiating bone marrow stem cells into dendritic cells. More specifically, bone marrow-derived DC (BMDC) was isolated from the femur and tibia of 6-8 week old female C57BL/6 mice, and then incubated with ACK lysis buffer (Lonza) to remove red blood cells. was processed. The cells were washed and cultured in a cell culture medium (RPMI 1640 containing 10% FBS and penicillin/streptomycin) containing 10 ng/ml mGM-CSF (Creagene Inc.). After 2 days, the supernatant of the cultured cells was removed and replaced with 2 ml of a new culture medium containing mGM-CSF.
- a cell culture medium RPMI 1640 containing 10% FBS and penicillin/streptomycin
- mature dendritic cells (mature DC, mDC) were prepared by culturing immature dendritic cells (immature DC) in the presence of LPS (100-200ng/ml) for 24 hours.
- BMDC was cultured after isolating monocytes from mouse bones to obtain dendritic cells. Using differentiated dendritic cells, each group was cultured for 1 hour after treatment according to the peptide concentration, and then each group was washed with PBS. In order to analyze the phenotype of dendritic cells, anti-mouse CD80, anti-mouse CD86, and anti-mouse CD11c were stained and measured using FACS CantoII, and analysis was performed with peptide-FITC to confirm peptide permeability in CD11c+. did
- a 100% TCA solution was added to the culture sample at a volume of 1/10, followed by strong vortexing, and then left in ice for 30 minutes, followed by centrifugation at 4°C and 14,000 rpm for 10 minutes. Thereafter, the supernatant was discarded, and 500 ul of cold acetone was added to the precipitate, followed by centrifugation at 4° C. and 14,000 rpm for 10 minutes, and then TCA was removed. Then, the precipitate was dried, resuspended using a buffer solution, and Western blotting was performed. As shown in FIG.
- BMDC cells were seeded in a 12-well plate at a number of 2 ⁇ 10 5 /ml, and then cultured at 37° C. for 1 day. Cells were treated with CTP-FITC, TAT-SEB2-FITC and CTP-SEB2-FITC peptides (Anigen Co., Ltd.) at a concentration of 1 uM attached to the cell surface for 4 hours, and the degree of fluorescence labeling of the cells was examined by FACS.
- BMDC was well prepared with a yield of 90% or more. It was confirmed that the effect was greater when SEB2 was grafted onto the CTP sequence owned by the company than the amount. This was also confirmed in the fusion protein results (FIG. 8).
- OVA (ovalbumin) antigen-specific TCR (OT-I) transgenic mice transgenic mice
- T cell proliferation response was analyzed through co-culture with DC using the mouse spleen.
- splenocytes R, OT-1 specific T cell,: 1X105 cells/well
- S OVA antigen-treated DCs
- R:S ratio ratio of 1:10 to 1:100
- the OVA-specific T cell response was the highest in the group containing DC sensitized with the peptide-fused antigen, and although the specific T-cell response increased in the case of CTP antigen-sensitized DC, it was higher than that of the peptide fusion antigen. It was confirmed that the reactivity was low.
- mice were immunized twice with dendritic cells sensitized with CTP-fusion melanoma recombinant antigen, and then A challenge test was conducted with a cancer cell line expressing a melanoma-specific antigen to examine whether solid cancer was formed.
- Mouse dendritic cells were used by differentiating bone marrow cells of the femur and tibia into dendritic cells. Both ends of the femur and tibia were cut, bone marrow cells were extracted, and the cells were collected in a 50 ml tube.
- the collected bone marrow cells were suspended in 0.83% ammonium chloride solution to remove red blood cells, and the bone marrow cells were dendritic cell production medium (RPMI-1640, 10% FBS, 10 ng/ml mouse recombinant IL-4 and 10 ng/ml mouse GM-CSF) for 2 days, and non-adherent cells were removed, and only cells attached to the bottom of the container were taken. Depletion of cytokines was prevented by replacing the culture medium with a fresh medium every 2-3 days. On day 6 of culture, immature dendritic cells were collected and treated with the recombinant antigens CTP-OVA and SCTP-OVA, respectively.
- RPMI-1640 10% FBS
- 10 ng/ml mouse recombinant IL-4 and 10 ng/ml mouse GM-CSF dendritic cell production medium
- Immature dendritic cells were sensitized by treating each antigenic protein at 10 ⁇ g/ml Korean Patent No. 10-0647847) for 20 hours, and cytokines necessary for dendritic cell maturation (100 ⁇ g/ml IFN- ⁇ and 100 ⁇ g/ml IFN- ⁇ ) were sensitized from 24 hours. ml TNF- ⁇ ) was added to induce maturation of dendritic cells. Anticancer immunity was induced by subcutaneously injecting dendritic cells 1x10 6 cells sensitized with the antigen into mice.
- Dendritic cell immunization was inoculated twice at 1-week intervals, and 1 week after the second dendritic cell inoculation, B16-OVA melanoma cells were SC (subcutaneous) injected into each 1x10 6 cell/mouse. Cancer size (width x height) was measured every 2 days. As a result of the measurement, the antitumor effect was confirmed as shown in FIG. 10 in the experimental group inoculated with dendritic cells sensitized with CTP-OVA or SCTP-OVA antigen.
- the permeability of the fusion peptide to be developed was observed in various immune cells (monocyte, T cell, B cell, Dendritic cell, NK cell, etc.) existing on PBMC. To do so, the frozen PBMC was thawed, and the CTP-SEB peptide fused with the existing CTP peptide was treated at a concentration of 1 uM, respectively, and analyzed by FACS. In the case of each immune cell, CD14 (monocyte), CD3 (T cell), CD19 (B cell), HLA-DR (dendritic cell), etc., which are representative markers of immune cells, were identified and the amount of permeated peptide was analyzed.
- the fusion peptide is permeable compared to the existing CTP peptide in monocyte, B cell, and dendritic cell known to express HLA-DR It was confirmed that the permeation amount was increased, and it was confirmed that the amount of permeation was additionally increased in T cells. Through this, it was confirmed that the permeation amount of the fusion peptide was increased in the immune cells as a whole.
- the polypeptide according to the present invention has an excellent ability to penetrate the cell membrane of immune cells, and can be effective in T cell proliferation by allowing antigens to be delivered with excellent efficiency through direct targeting to immune cells.
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Abstract
The present invention relates to a polypeptide for delivering an antigen to immune cells and, specifically, to: a novel polypeptide including a cell membrane penetrating peptide and a peptide binding to a surface molecule on immune cells; a fusion polypeptide in which an antigen is coupled to the polypeptide; a nucleic acid coding for the polypeptide or the fusion polypeptide; an immune cell immunized with the fusion polypeptide or the nucleic acid coding therefor; and an immunotherapeutic agent, antitumor or anticancer vaccine, and a composition for treating a tumor or cancer, each comprising the immune cell.
Description
본 발명은 면역세포에 항원을 전달하기 위한 폴리펩타이드에 관한 것으로, 구체적으로 세포막 투과 펩타이드 및 면역세포의 표면 분자에 결합하는 펩타이드를 포함하는 신규 폴리펩타이드, 상기 폴리펩타이드에 항원이 결합되어 있는 융합 폴리펩타이드, 상기 폴리펩타이드 또는 융합 폴리펩타이드를 코딩하는 핵산, 상기 융합 폴리펩타이드 또는 이를 코딩하는 핵산에 의해 감작된 면역세포, 면역세포를 포함하는 면역치료제, 항-종양 또는 항암 백신 및 종양 또는 암 치료용 조성물에 관한 것이다. The present invention relates to polypeptides for delivering antigens to immune cells, and specifically, novel polypeptides including cell membrane penetrating peptides and peptides that bind to surface molecules of immune cells, and fusion polypeptides to which antigens are bound to the polypeptides. Peptides, nucleic acids encoding said polypeptides or fusion polypeptides, immune cells sensitized by said fusion polypeptides or nucleic acids encoding them, immunotherapeutic agents comprising immune cells, anti-tumor or anti-cancer vaccines and for treatment of tumors or cancers It's about the composition.
FGF (Fibroblast Growth Factor)에 존재하는 시그널 서열 (막투과 서열: membrane transduction sequence: MTS) 또는 세포질 침투 펩타이드 (cytoplasmic penetration peptide: CPP)는 세포막을 투과하는 성질을 가지는 것으로 알려졌다. 상기 시그널 서열은 세포막을 쉽게 투과하는 성질을 가지나, NLS (nuclear localization sequence)가 없어서 핵 내로는 들어가지 않는 특징을 보인다.Signal sequences (membrane transduction sequence: MTS) or cytoplasmic penetration peptide (CPP) present in FGF (Fibroblast Growth Factor) are known to have the property of penetrating cell membranes. The signal sequence has a property of easily penetrating the cell membrane, but does not enter the nucleus because it does not have a nuclear localization sequence (NLS).
MTS 또는 CPP는 항원 전달체로 사용될 수 있다. 세포질 내로 유입된 펩타이드 항원이 프로테아좀 (proteasome)을 거쳐 프로세싱 되면서 MHC 클래스 I에 실려 세포 표면으로 제시될 수 있다. 또한, 세포질에서 진행되는 세포 죽음과 세포 성장을 조절하는 여러 신호 전달 과정을 대상으로 하여 개발된 특정 기능의 단백질을 전달하기 위한 치료용 물질 전달에 사용될 수 있다. MTS or CPP can be used as an antigen carrier. Peptide antigens introduced into the cytoplasm are processed through the proteasome and presented on the cell surface on MHC class I. In addition, it can be used for therapeutic substance delivery to deliver proteins with specific functions developed targeting various signal transduction processes that control cell death and cell growth in the cytoplasm.
이와 관련하여, 본 출원의 발명자들은 세포막 투과능은 있으면서도 세포질에 잔류하는, 세포질 잔류성 세포막 투과 펩타이드 (cytoplasmic transduction peptide, CTP)를 개발하고, CTP가 우수한 세포막 투과능을 보이면서도 세포질 내에 잔류하는 신규한 특성을 보유하고 있기 때문에, 세포 독성 T 림프구 (cytotoxic T lymphocyte; CTL)-반응을 유도하는 데 적합하며, 세포질 내로의 효과적인 약물송달시스템 (drug delivery system; DDS)으로 사용될 수 있음을 확인한 바 있다 (한국등록특허 제0608558호).In this regard, the inventors of the present application develop a cytoplasmic transduction peptide (CTP) that remains in the cytoplasm while having cell membrane penetrating ability, and develops a novel CTP that remains in the cytoplasm while showing excellent cell membrane penetrating ability. Since it has properties, it has been confirmed that it is suitable for inducing cytotoxic T lymphocyte (CTL)-reaction and can be used as an effective drug delivery system (DDS) into the cytoplasm ( Korean Patent Registration No. 0608558).
한편, 황색포도알균 (Staphylococcus aureus)은 자연환경에 대한 저항성이 강하기 때문에 자연계에 광범위하게 분포하고 있으며, 사람이나 동물의 화농성 변소에 존재할 뿐만 아니라 건강한 사람과 동물의 피부 등에도 상재하고 있어 식품과 인체에 오염될 가능성이 매우 높다. 황색포도알균 (Staphylococcus aureus)이 생성하는 병원성 인자는 외독소 (exotoxin), 장독소 (enterotoxin), 세포 용해성 독소 (hemolysin toxin), 백혈구 사멸독소 (leucodicin), 피부용해 독소 (epidermolytic toxin) 이외에 독소성 쇼크 증후군 독소 (toxin shock syndrome toxin, TSST) 등의 세포외 산물을 생성함으로써, 인간에게 식중독 등의 중요한 각종 질병을 유발한다.On the other hand, Staphylococcus aureus is widely distributed in the natural world because of its strong resistance to the natural environment. are very likely to be contaminated. The pathogenic factors produced by Staphylococcus aureus include exotoxin, enterotoxin, hemolysin toxin, leucodicin, and epidermolytic toxin, as well as toxic shock. By producing extracellular products such as toxin shock syndrome toxin (TSST), it causes various important diseases such as food poisoning in humans.
황색포도알균 장독소 B형 (Staphylococcal Enterotoxin B 또는 이하 'SEB'라고도 함)는 황색포도알균 (Staphylococcus aureus) 독소형 식중독균에 의해 생산되는 독소로서, 전 세계적으로 분포되어 있고 주로 식중독을 일으키고 비정상적인 면역반응을 유도하여 심각한 면역질환을 일으키는 원인이 된다.Staphylococcal enterotoxin B type (Staphylococcal Enterotoxin B or hereinafter referred to as 'SEB') is a toxin produced by a toxin-type food poisoning bacterium of Staphylococcus aureus , which is distributed worldwide and mainly causes food poisoning and abnormal immune response. can lead to serious autoimmune diseases.
SEB는 약 26 내지 29 kDa 크기의 단백질로써 분자량은 작으나 SEB는 물에 잘 녹고 펩신(pepsin), 트립신(trypsin), 폐폐인(papain) 등과 같은 프로테아제(Protease) 효소에 대한 저항성도 강하며 극한 온도나 pH에도 잘 견디는 매우 안정한 독소이다. SEB is a protein of about 26 to 29 kDa in size and has a small molecular weight, but SEB is highly soluble in water, has strong resistance to protease enzymes such as pepsin, trypsin, and papain, and is resistant to extreme temperatures. It is a very stable toxin that tolerates pH well.
SEB의 기능적 측면에서, SEB는 슈퍼항원(Superantigen, SAg)으로 명명되며 활성화된 CD4+ 또는 CD8+ T 세포에 존재하는 T-세포 수용체(T-cell receptor, TCR)와 항원표출세포(Antigen Presenting Cell, APC)에 존재하는 MHC 클래스 Ⅱ(Major Histocompatibility Complex class II) 사이를 연결하는 역할을 하는 항원(antigen)이다. SEB는 대식세포의 MHC 클래스 Ⅱ에 결합한 후, T 세포의 수용체 Vb8 부위와도 결합함으로써, T세포를 비정상적으로 활성화시켜, IL-6, IL-1β, MIP-1 (Macrophage Inflammatory Protein-1) 등과 같은 다량의 염증성 사이토카인을 대량으로 분비되도록 함으로써, 비정상적인 면역반응을 유도하게 된다. 호흡기계가 SEB에 노출되었을 때, SEB로 사망하는 사람은 적지만, 노출된 사람의 80% 이상은 임상적 증상을 나타내고, 1~2주 동안은 일상생활이 불가능할 정도로 심한 증상을 나타내는 것으로 알려졌다. In terms of the function of SEB, SEB is named superantigen (SAg), and is associated with the T-cell receptor (TCR) present on activated CD4+ or CD8+ T cells and Antigen Presenting Cell (APC). It is an antigen that plays a role in linking between MHC class II (Major Histocompatibility Complex class II) present in ). SEB binds to MHC class II of macrophages and then binds to the receptor Vb8 of T cells, thereby abnormally activating T cells and producing IL-6, IL-1β, MIP-1 (Macrophage Inflammatory Protein-1), etc. By secreting a large amount of the same amount of inflammatory cytokines, an abnormal immune response is induced. When the respiratory system is exposed to SEB, there are few people who die of SEB, but more than 80% of exposed people show clinical symptoms, and it is known that they show symptoms that are so severe that daily life is impossible for 1 to 2 weeks.
이러한 기술적 배경하에서, 본 출원의 발명자들은 세포막 투과 펩타이드 및 SEB의 구조적 연구를 통해, MHC 클래스 Ⅱ에 결합하는 펩타이드를 선별하였으며, 세포막 투과 펩타이드 및 면역세포의 표면 분자에 결합하는 펩타이드를 포함하는 신규 폴리펩타이드가 면역세포로의 항원 전달 수율을 향상시킬 수 있음을 확인하고, 본 발명을 완성하였다. Under such a technical background, the inventors of the present application have selected peptides that bind to MHC class II through structural studies of cell membrane penetrating peptides and SEB, and a novel polytype including cell membrane penetrating peptides and peptides that bind to surface molecules of immune cells It was confirmed that the peptide can improve the yield of antigen delivery to immune cells, and the present invention was completed.
발명의 요약Summary of Invention
본 발명의 목적은 면역세포에 항원을 전달하기 위한 신규 폴리펩타이드를 제공하는데 있다. An object of the present invention is to provide novel polypeptides for delivering antigens to immune cells.
본 발명의 목적은 상기 폴리펩타이드를 코딩하는 핵산을 제공하는데 있다.An object of the present invention is to provide a nucleic acid encoding the polypeptide.
본 발명의 목적은 상기 폴리펩타이드에 항원이 결합되어 있는 융합 폴리펩타이드를 제공하는데 있다.An object of the present invention is to provide a fusion polypeptide in which an antigen is bound to the polypeptide.
본 발명의 목적은 상기 융합 폴리펩타이드를 코딩하는 핵산을 제공하는데 있다.An object of the present invention is to provide a nucleic acid encoding the fusion polypeptide.
본 발명의 목적은 상기 융합 폴리펩타이드 또는 상기 핵산을 포함하는 면역세포를 제공하는데 있다.An object of the present invention is to provide an immune cell containing the fusion polypeptide or the nucleic acid.
본 발명의 목적은 상기 면역세포를 포함하는 면역치료제를 제공하는데 있다.An object of the present invention is to provide an immunotherapeutic agent comprising the immune cells.
본 발명의 목적은 상기 면역세포를 포함하는 항-종양 또는 항암 백신을 제공하는데 있다.An object of the present invention is to provide an anti-tumor or anti-cancer vaccine comprising the immune cells.
본 발명의 목적은 상기 면역세포를 포함하는 종양 또는 암 치료용 조성물을 제공하는데 있다. 본 발명의 목적은 상기 면역세포를 개체에 투여하는 것을 포함하는 종양 또는 암 치료방법을 제공하는데 있다. 본 발명의 목적은 상기 면역세포를 종양 또는 암 치료용 조성물 제조에 사용하는 용도를 제공하는데 있다.An object of the present invention is to provide a composition for treating tumor or cancer containing the immune cells. An object of the present invention is to provide a method for treating tumor or cancer comprising administering the immune cells to a subject. An object of the present invention is to provide a use of the immune cells for preparing a composition for treating tumor or cancer.
상기 목적을 달성하기 위하여, 본 발명은 세포막 투과 펩타이드 (cytoplasmic transduction peptide, CTP) 및 면역세포의 표면 분자에 결합하는 펩타이드를 포함하는, 면역세포에 항원을 전달하기 위한 폴리펩타이드를 제공한다.In order to achieve the above object, the present invention provides a polypeptide for delivering an antigen to immune cells, including a cytoplasmic transduction peptide (CTP) and a peptide that binds to surface molecules of immune cells.
본 발명은 상기 폴리펩타이드를 코딩하는 핵산을 제공한다.The present invention provides nucleic acids encoding the above polypeptides.
본 발명은 상기 폴리펩타이드에 항원이 결합되어 있는 융합 폴리펩타이드를 제공한다.The present invention provides a fusion polypeptide in which an antigen is bound to the polypeptide.
본 발명은 상기 융합 폴리펩타이드를 코딩하는 핵산을 제공한다.The present invention provides nucleic acids encoding the fusion polypeptides.
본 발명은 상기 융합 폴리펩타이드 또는 상기 융합 폴리펩타이드를 코딩하는 핵산을 포함하는 면역세포를 제공한다.The present invention provides an immune cell containing the fusion polypeptide or a nucleic acid encoding the fusion polypeptide.
본 발명은 상기 면역세포를 포함하는 면역치료제를 제공한다.The present invention provides an immunotherapeutic agent comprising the above immune cells.
본 발명은 상기 면역세포를 포함하는 항-종양 또는 항암 백신을 제공한다.The present invention provides an anti-tumor or anti-cancer vaccine comprising the above immune cells.
본 발명은 상기 면역세포를 포함하는 종양 또는 암 치료용 조성물을 제공한다. 본 발명은 상기 면역세포를 개체에 투여하는 것을 포함하는 종양 또는 암 치료방법을 제공한다. 본 발명은 상기 면역세포를 종양 또는 암 치료용 조성물 제조에 사용하는 용도를 제공한다.The present invention provides a composition for treating tumor or cancer comprising the immune cells. The present invention provides a method for treating tumor or cancer comprising administering the immune cells to a subject. The present invention provides a use of the immune cells for preparing a composition for treating tumor or cancer.
도 1은 CTP/CTP-SEB 그룹의 MoDC 세포막 투과성을 비교한 결과를 나타낸 것이다.Figure 1 shows the results of comparing the MoDC cell membrane permeability of the CTP / CTP-SEB group.
도 2는 CTP/CTP-SEB 그룹의 MoDC 투과성 차이를 나타낸 것이다.Figure 2 shows the difference in MoDC permeability of the CTP/CTP-SEB group.
도 3은 재조합 융합단백질 생산을 위한 벡터를 나타낸 것이다. 3 shows a vector for producing a recombinant fusion protein.
도 4는 재조합 융합단백질 생산에서 Ni-NTA 레진을 이용한 정제 결과 및 QC분석 결과를 나타낸 것이다.Figure 4 shows the results of purification and QC analysis using Ni-NTA resin in the production of recombinant fusion proteins.
도 5는 융합단백질의 세포투과성 측정 결과를 나타낸 것이다.5 shows the results of measuring cell permeability of fusion proteins.
도 6은 융합단백질의 직접적인 결합력 분석 결과를 나타낸 것이다.6 shows the results of direct avidity analysis of fusion proteins.
도 7은 배양액 내 단백질 잔존량 분석 결과를 나타낸 것이다.Figure 7 shows the results of analysis of the remaining amount of protein in the culture medium.
도 8은 PTD 계열 펩타이드의 DC 표현형 및 세포막 투과성 비교 실험 결과를 나타낸 것이다.Figure 8 shows the results of DC phenotype and cell membrane permeability comparison experiment of PTD series peptides.
도 9는 항원 특이적 T cell 증식 분석 결과를 나타낸 것이다.9 shows the results of antigen-specific T cell proliferation assay.
도 10은 CTP-OVA 또는 SCTP-OVA 항원으로 감작된 수지상세포를 접종시킨 마우스에서 항종양 효과를 확인한 결과를 나타낸 것이다. Figure 10 shows the results of confirming the antitumor effect in mice inoculated with dendritic cells sensitized with CTP-OVA or SCTP-OVA antigen.
도 11은 면역세포 종류에 따른 투과성 분석 결과를 나타낸 것이다. 11 shows the results of permeability analysis according to immune cell types.
발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.
일 관점에서 본 발명은 세포막 투과 펩타이드 (cytoplasmic transduction peptide, CTP) 및 면역세포의 표면 분자에 결합하는 펩타이드를 포함하는, 면역세포에 항원을 전달하기 위한 폴리펩타이드에 관한 것이다. In one aspect, the present invention relates to a polypeptide for delivering an antigen to immune cells, including a cytoplasmic transduction peptide (CTP) and a peptide that binds to surface molecules of immune cells.
상기 세포막 투과 펩타이드는 예를 들어, 단백질 운반 도메인 예를 들어, CTP(cytoplasmic transduction peptide, 세포질 잔류성 세포막 투과 펩타이드), HP4, Hph-1, Mph-1, Sim-2, Tat, VP22, Antp(Antennapedia), Pep-1(peptide), PTD-5, R9(arginine) 및 7R 도메인을 포함하는 펩타이드로 구성된 군에서 선택된 하나 이상일 수 있으나, 이에 제한되는 것은 아니다. The cell membrane penetrating peptide is, for example, a protein transduction domain, for example, CTP (cytoplasmic transduction peptide, cytoplasmic residual cell membrane penetrating peptide), HP4, Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia ), Pep-1 (peptide), PTD-5, R9 (arginine), and may be one or more selected from the group consisting of peptides containing the 7R domain, but is not limited thereto.
구체적으로, 상기 세포막 투과 펩타이드는 세포막 투과능을 갖으며, 세포에 처리하고 일정 시간 후 상기 처리된 세포에 단백질 분해효소를 처리한 경우에도 세포막 투과현상을 나타내고, 세포막을 투과한 이후에는 세포질에 잔류하는 특성을 갖는 세포질 잔류성 세포막 투과 펩타이드일 수 있다. 상기 세포막 투과 펩타이드가 면역세포의 표면 분자에 결합하는 펩타이드와 융합시, 엔도사이토시스 (endocytosis)를 억제함과 동시에 유비퀴틴 (ubiquitin)을 억제하는 경우 폴리펩타이드가 분해되지 않고 세포 내 존재하는 것을 확인하였다. Specifically, the cell membrane penetrating peptide has cell membrane penetrating ability, shows cell membrane penetrating phenomenon even when a cell is treated with a proteolytic enzyme after a certain period of time, and remains in the cytoplasm after penetrating the cell membrane It may be a cytoplasmic residual cell membrane penetrating peptide having the characteristics of When the cell membrane penetrating peptide is fused with a peptide that binds to a surface molecule of an immune cell, endocytosis is inhibited and ubiquitin is inhibited at the same time. It was confirmed that the polypeptide is not degraded and exists in the cell .
상기 세포막 투과 펩타이드는 예를 들어, 9-20 aa, 보다 바람직하게는 9-15 aa, 가장 바람직하게는 약 11 aa 길이를 가질 수 있다. The cell membrane penetrating peptide may have a length of, for example, 9-20 aa, more preferably 9-15 aa, and most preferably about 11 aa.
상기 세포막 투과 펩타이드는 예를 들어, 상기 세포막 투과 펩타이드는 서열번호 5 내지 7로 구성된 군에서 선택되는 서열을 포함할 수 있다. 본 출원의 발명자들은 서열번호 5 내지 7로 구성된 군에서 선택되는 서열을 포함하는 세포막 투과 펩타이드를 면역세포의 표면 분자에 결합하는 펩타이드와 융합하는 경우, 세포막 투과 펩타이드 단독에 비해 우수한 세포막 투과능을 나타낼 수 있음을 확인하였다. The cell membrane penetrating peptide may include, for example, a sequence selected from the group consisting of SEQ ID NOs: 5 to 7. The inventors of the present application found that when a cell membrane penetrating peptide containing a sequence selected from the group consisting of SEQ ID NOs: 5 to 7 is fused with a peptide that binds to a surface molecule of an immune cell, it exhibits superior cell membrane penetrating ability compared to the cell membrane penetrating peptide alone. confirmed that it can.
상기 면역세포의 표면 분자에 결합하는 펩타이드는 예를 들어, MHC (major histocompatibility complex) 분자에 결합하는 펩타이드일 수 있다. 구체적으로, 상기 면역세포의 표면 분자로 발현하는 MHC (major histocompatibility complex) 클래스 II (MHC class II)에 결합하는 펩타이드를 포함할 수 있다. The peptide that binds to the surface molecule of the immune cell may be, for example, a peptide that binds to a major histocompatibility complex (MHC) molecule. Specifically, it may include a peptide that binds to major histocompatibility complex (MHC) class II expressed as surface molecules of the immune cells.
하나의 구체예에서, 상기 면역세포의 표면 분자에 결합하는 펩타이드는 서열번호 1 내지 4로 구성된 군에서 선택되는 서열을 포함할 수 있다. In one embodiment, the peptide binding to the surface molecule of the immune cell may include a sequence selected from the group consisting of SEQ ID NOs: 1 to 4.
상기 항원은 암 또는 종양에 대한 면역반응 활성화를 위한 암 또는 종양 항원일 수 있다. 상기 항원은 암 또는 종양 항원으로 세포치료에 있어 암 또는 종양 특이적 면역세포 예를 들어, 세포독성 T 세포를 유도할 수 있다. 상기 암 또는 종양 항원은 예를 들어, 자가(self) 또는 비자가(non-self) 유래 항원일 수 있다. 예를 들면 환자 자신의 유전자에서 유래된 자가 암 또는 종양 항원은 hTERT (GenBank: BAC11010.1), WT1 (GenBank: AAO61088.1), NY-ESO1 (GenBank: CAA05908.1), MAGE-A3 (NCBI Reference Sequence: NP_005353.1) 등과 암 특이적 돌연변이 항원 예를 들면 neoantigens, mutated P53, RAS 등의 종양 억제 또는 유발 유전자를 포함할 수 있으며, 외래 암 또는 종양 항원으로 암 또는 종양 유발 바이러스 항원 예를 들면 CMV, EBV, HPV 등을 포함할 수 있다.The antigen may be a cancer or tumor antigen for activating an immune response against cancer or tumor. The antigen is a cancer or tumor antigen and can induce cancer or tumor specific immune cells, such as cytotoxic T cells, in cell therapy. The cancer or tumor antigen may be, for example, a self or non-self derived antigen. For example, autologous cancer or tumor antigens derived from a patient's own gene include hTERT (GenBank: BAC11010.1), WT1 (GenBank: AAO61088.1), NY-ESO1 (GenBank: CAA05908.1), MAGE-A3 (NCBI Reference Sequence: NP_005353.1) and cancer-specific mutated antigens, such as neoantigens, mutated P53, and RAS, may include tumor suppressor or inducing genes, and cancer or tumor-inducing virus antigens such as foreign cancer or tumor antigens CMV, EBV, HPV, and the like.
자가 암 또는 종양 항원인 hTERT는 염색체 말단에서 텔로미어 DNA(telomeric DNA)를 합성하는 효소로서 암세포는 이 효소를 과도하게 활성화시켜 텔로미어 의존적 세포사멸을 회피할 수 있도록 기능하고 폐암, 위암, 췌장암을 포함하는 다양한 고형암의 타겟 항원으로 알려져 있고 (Kim NW, et al. Science. 1994;266:2011-2015), WT1은 Wilms tumor와 관련된 유전자로서 징크 핑거 전사인자를 암호화하여 세포의 증식과 분화, 자멸사, 기관의 발생에 관여를 하는 단백질로서 뇌척수암, 폐암 등의 타겟 항원으로 알려져 있다 (Call KM, et al., Cell. 1990. 60:509-520; Nakahara Y, et al.,Brain Tumor Pathol. 2004. 21:113-6). 또한 NY-ESO1은 CTA (cancer testis antigen)에 속하는 단백질 중 하나로 주로 생식세포(germ cell)와 육종(sarcoma), 유방암을 포함한 다양한 암세포에 발현하는 것으로 잘 알려져 있다 (Gnjatic S, et al., Adv Cancer Res. 2006;95:1-30). MAGE-A3은 멜라노마 연관 항원 패밀리 (melanoma-associated antigen family)에 속하는 단백질로 폐암, 육종 및 흑색종을 포함한 다양한 암세포에 과발현하는 것으로 알려져 있어 암의 면역치료에 적합한 표적 항원으로 평가되고 있다 (Decoster L, et al.,Ann Oncol. 2012 Jun;23(6):1387-93).hTERT, an autologous cancer or tumor antigen, is an enzyme that synthesizes telomeric DNA at the ends of chromosomes, and cancer cells activate this enzyme excessively to avoid telomere-dependent apoptosis. It is known as a target antigen for various solid cancers (Kim NW, et al. Science. 1994;266:2011-2015), and WT1 is a gene related to Wilms tumor that encodes a zinc finger transcription factor, which causes cell proliferation and differentiation, apoptosis, and organs. As a protein involved in the development of cerebrospinal cancer, it is known as a target antigen for lung cancer, etc. (Call KM, et al., Cell. 1990. 60:509-520; Nakahara Y, et al., Brain Tumor Pathol. 2004. 21:113-6). In addition, NY-ESO1 is one of the proteins belonging to CTA (cancer testis antigen) and is well known to be expressed in various cancer cells, including germ cells, sarcoma, and breast cancer (Gnjatic S, et al., Adv. Cancer Res. 2006;95:1-30). MAGE-A3 is a protein belonging to the melanoma-associated antigen family and is known to be overexpressed in various cancer cells, including lung cancer, sarcoma, and melanoma, and is being evaluated as a suitable target antigen for cancer immunotherapy (Decoster L, et al., Ann Oncol. 2012 Jun;23(6):1387-93).
경우에 따라서, 상기 항원은 예를 들어, A33, ALK, 알파페토단백질(AFP), 아드레날린 수용체 베타 3(ADRB3), alpha-folate receptor, AD034, AKT1, BCMA, 베타-인간 융모막 고나도트로핀, B7H3(CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD79a, CD79b, CD89, CD97, CD123, CD138, CD160, CD171, CD179a, 탄산무수화효소 IX(CAIX), CA-125, 발암배아성 항원(CEA), CCR4, C-유형 렉틴-유사 분자-1(CLL-1 또는 CLECL1), 클라우딘 6(CLDN6), CXORF61, CAGE, CDX2, CLP, CT-7, CT8/HOM-TES-85, cTAGE-1, ERBB2, 상피 성장 인자 수용체(EGFR), EGFR 변이체 III(EGFRvIII), 상피 세포 부착 분자(EPCAM), 신장 인자 2 돌연변이된(ELF2M), 에프린 유형-A 수용체 2(EphA2), EMR2, Fms-유사 티로신 키나제 3(FLT3), FCRL5, Fibulin-1, G250, GD2, 당단백질 36(gp36), 당단백질 100(gp100), 글루코코르티코이드-유도된 종양 괴사 인자 수용체(GITR), GPRC5D, GloboH, G 단백질-결합된 수용체 20(GPR20), GPC3, hsp70-2, 고분자량-흑색종-관련 항원(HMWMAA), A형 간염 바이러스 세포 수용체 1(HAVCR1), HAGE, HCA587/MAGE-C2, hCAP-G, HCE661, HER2/neu, HLA-Cw, HOM-HD-21/Galectin9, HOM-MEEL- 40/SSX2, HOM-RCC-3.1.3/CAXII, HOXA7, HOXB6, Hu, HUB 1, 인슐린 성장 인자(IGF1)-I, IGF-II, IGFI 수용체, 인터류킨-13 수용체 서브유닛 알파-2(IL-13Ra2 또는 CD213A2), 인터류킨 11 수용체 알파(IL-11Ra), IGLL1, KIT(CD117), KM-HN-3, KM-KN- 1, KOC1, KOC2, KOC3, LAGA-1a, LAGE-1, LAIR1, LILRA2, LY75, Lewis Y antigen, MUC1, MN-CA IX, M-CSF, MAGE-1, MAGE-4a, 메소텔린, MAGE-A1, MAD-CT-1, MAD-CT-2, MART1, MPPl 1, MSLN, 신경세포 부착 분자(NCAM), NY-ESO-1, NY-ESO-5, Nkp30, NKG2D, 유선 분화 항원(NY-BR-1), NY-BR-62, NY-BR-85, NY-CO-37, NY-CO-38, NNP-1, NY-LU-12, NY-REN-10, NY-REN-19/LKB/STK1 1, NY-REN-21, NY-REN-26/BCR, NY-REN-3/NY-CO-38, NY-REN-33/SNC6, NY-REN-43, NY-REN-65, NY-REN-9, NY-SAR-35, o-아세틸-GD2 강글리오시드(OAcGD2), OGFr, PSMA, 전립선 산성 포스파타제(PAP), p53, 전립선-암종 종양 항원-1(PCTA-1), 전립선 줄기세포 항원(PSCA), 프로테아제세린 21(테스티신 또는 PRSS21), 혈소판-유래된 성장인자 수용체 베타(PDGFR-beta), PLAC1, 파넥신 3(PANX3), PLU-1, ROR-1, RAGE-1, RU1, RU2, Rab38, RBPJkappa, RHAMM, 단계-특이적 배아 항원-4(SSEA-4), SCP1, SSX3, SSX4, SSX5, Tyrp-1, TAG72, 티로글로불린, 5T4, 종양-관련 당단백질 72(TAG72), 티로시나제(Tyrosinase), 트랜스글루타미나제 5(TGS5), TEM1, TEM7R, 갑상선 자극 호르몬 수용체(TSHR), Tie 2, TRP-2, TOP2A, TOP2B, 유로플라킨 2(UPK2), 비멘틴(Vimentin), 또는 혈관 내피성장 인자 수용체 2(VEGFR2)일 수 있다. Optionally, the antigen is, for example, A33, ALK, alpha-fetoprotein (AFP), adrenergic receptor beta 3 (ADRB3), alpha-folate receptor, AD034, AKT1, BCMA, beta-human chorionic gonadotropin, B7H3 (CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD79a, CD79b, CD89, CD97, CD123, CD138, CD160, CD171 , CD179a, carbonic anhydrase IX (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR4, C-type lectin-like molecule-1 (CLL-1 or CLECL1), claudin 6 (CLDN6), CXORF61 , CAGE, CDX2, CLP, CT-7, CT8/HOM-TES-85, cTAGE-1, ERBB2, epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), epithelial cell adhesion molecule (EPCAM), elongation factor 2 Mutated (ELF2M), Ephrin Type-A Receptor 2 (EphA2), EMR2, Fms-Like Tyrosine Kinase 3 (FLT3), FCRL5, Fibulin-1, G250, GD2, Glycoprotein 36 (gp36), Glycoprotein 100 (gp100), glucocorticoid-induced tumor necrosis factor receptor (GITR), GPRC5D, GloboH, G protein-coupled receptor 20 (GPR20), GPC3, hsp70-2, high molecular weight-melanoma-associated antigen (HMWMAA), Hepatitis A virus cellular receptor 1 (HAVCR1), HAGE, HCA587/MAGE-C2, hCAP-G, HCE661, HER2/neu, HLA-Cw, HOM-HD-21/Galectin9, HOM-MEEL-40/SSX2, HOM -RCC-3.1.3/CAXII, HOXA7, HOXB6, Hu, HUB 1, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2) , interleukin 11 receptor alpha (IL-11Ra), IGLL1, KIT (CD117), KM-HN-3, KM-KN-1, KOC1, KOC2, KOC3, LAGA-1a, LAGE-1, LAIR1, LILRA2, LY75, Lewis Y antigen, MUC1, MN-CA IX, M-CSF, MAGE-1, MAGE-4a, mesothelin, MAGE-A1, MAD-CT-1, MAD-CT-2, MART1, MPPl 1, MSLN, neuron Cell Adhesion Molecule (NCAM), NY-ESO-1, NY-ESO-5, Nkp30, NKG2D, Mammary Differentiation Antigen (NY-BR-1), NY-BR-62, NY-BR-85, NY-CO- 37, NY-CO-38, NNP-1, NY-LU-12, NY-REN-10, NY-REN-19/LKB/STK1 1, NY-REN-21, NY-REN-26/BCR, NY -REN-3/NY-CO-38, NY-REN-33/SNC6, NY-REN-43, NY-REN-65, NY-REN-9, NY-SAR-35, o-acetyl-GD2 ganglio seed (OAcGD2), OGFr, PSMA, prostate acid phosphatase (PAP), p53, prostate-carcinoma tumor antigen-1 (PCTA-1), prostate stem cell antigen (PSCA), protease serine 21 (testicine or PRSS21), platelets -derived growth factor receptor beta (PDGFR-beta), PLAC1, panexin 3 (PANX3), PLU-1, ROR-1, RAGE-1, RU1, RU2, Rab38, RBPJkappa, RHAMM, stage-specific embryonic antigens -4 (SSEA-4), SCP1, SSX3, SSX4, SSX5, Tyrp-1, TAG72, thyroglobulin, 5T4, tumor-associated glycoprotein 72 (TAG72), tyrosinase, transglutaminase 5 (TGS5) ), TEM1, TEM7R, Thyroid Stimulating Hormone Receptor (TSHR), Tie 2, TRP-2, TOP2A, TOP2B, Europlakin 2 (UPK2), Vimentin, or Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) can
또한, 상기 항원은 병원균 (박테리아, 바이러스 등), 화합물질, 꽃가루, 암세포, 새우 등 또는 이들의 일부 펩타이드 또는 단백질이며, 더욱 바람직하게는 암 항원 펩타이드이나, 체내에서 면역 반응을 일으킬 수 있는 물질이라면 이에 제한되지 않는다. 상기 항원은 바람직하게는 단백질, 재조합 단백질, 당단백질, 유전자, 펩티드, 다당류, 지질다당류, 폴리뉴클레오티드, 세포, 세포 용해물(lysate), 박테리아, 바이러스 등일 수 있으며, 더욱 바람직하게는, 암 항원 펩타이드일 수 있다. 상기 당백질은 항체, 항체의 단편, 구조 단백질, 조절 단백질, 전사인자, 독소 단백질, 호르몬, 호르몬 유사체, 효소, 효소의 단편, 수송 단백질, 수용체(receptor), 수용체의 단편, 생체방어 유도 물질, 저장 단백질, 이동 단백질(movement protein), 익스플로이티브 단백질(exploitive protein), 리포터 단백질 등일 수 있다. 그러나 생체에서 항원으로 작용하여 면역 반응을 유도할 수 있는 물질이라면 이에 제한되지 않는다.In addition, the antigen is a pathogen (bacterium, virus, etc.), a chemical substance, pollen, cancer cell, shrimp, etc., or some peptide or protein thereof, more preferably a cancer antigen peptide, or a substance that can cause an immune response in the body Not limited to this. The antigen may preferably be a protein, recombinant protein, glycoprotein, gene, peptide, polysaccharide, lipopolysaccharide, polynucleotide, cell, cell lysate, bacterium, virus, etc., more preferably, a cancer antigen peptide can be The protein includes antibodies, antibody fragments, structural proteins, regulatory proteins, transcription factors, toxin proteins, hormones, hormone analogues, enzymes, enzyme fragments, transport proteins, receptors, fragments of receptors, biological defense inducers, It may be a storage protein, a movement protein, an exploitive protein, a reporter protein, and the like. However, any material capable of inducing an immune response by acting as an antigen in a living body is not limited thereto.
본 발명에 따른 항원은 본 발명에 따른 폴리펩타이드와 결합할 수 있는 항원으로, 불활성화 종양세포, 유전자 재조합 방법에 의해 제조된 종양세포 관련 유전자, 펩타이드 또는 단백질을 포함할 수 있다. 유전자 재조합 방법에 의해 상기 항원을 수득하고자 하는 경우, 상기 항원을 인코딩하는 뉴클레오타이드 서열은 공지일 수 있으며, 공지된 서열의 전장을 이용할 수 있으나, 전장의 일부를 이용할 수도 있다. 상기 항원을 인코딩하는 뉴클레오타이드 서열을 벡터에 클로닝하여 목적하는 항원이 발현되도록 할 수 있다.The antigen according to the present invention is an antigen capable of binding to the polypeptide according to the present invention, and may include inactivated tumor cells, tumor cell-related genes, peptides or proteins produced by genetic recombination. When obtaining the antigen by genetic recombination, the nucleotide sequence encoding the antigen may be known, and the full length of the known sequence may be used, but a part of the full length may also be used. A nucleotide sequence encoding the antigen may be cloned into a vector to express the desired antigen.
상기 항원은 폴리펩타이드의 N-말단 또는 C-말단에 결합할 수 있다. 공유결합 방법은 항원의 종류에 따라 당업계에 공지된 방법을 이용하여 실시할 수 있으며, 예를 들어 폴리펩타이드를 인코딩하는 유전자를 클로닝 및 세포 내 발현시켜 수득할 수 있다.The antigen may bind to the N-terminus or C-terminus of the polypeptide. The covalent bonding method may be performed using a method known in the art depending on the type of antigen, and may be obtained, for example, by cloning and intracellularly expressing a gene encoding a polypeptide.
경우에 따라서, 폴리펩타이드의 활성을 방해하지 않는 링커, 예를 들어 N-숙시니미딜 요오도아세테이드, N-말레이미도부티릴 옥시숙신아미드 에스테르, 1,5-디플루오로-2,4-디니트로벤젠, 디스디아조벤지딘, 3,3-디티오-비스-(설포숙시니미딜-프로피오네이트), 에틸렌 글리콜비스(설포숙시니미딜숙시네이트), 디시클로헥실 카보디이미드 등이 이용될 수 있으나, 이에 한정되지 아니한다. 한편, 항원이 폴리펩타이드로부터 분해되었을 때에만 활성을 나타내는 경우에는, 링커는 생체 내에서 절단 가능한 것을 사용한다. 예를 들어, 카복실산 에스테르 및/또는 디설파이드 결합이 있는 결합제가 이용될 수 있다.Optionally, a linker that does not interfere with the activity of the polypeptide, for example N-succinimidyl iodoacetate, N-maleimidobutyryl oxysuccinamide ester, 1,5-difluoro-2,4 -Dinitrobenzene, disdiazobenzidine, 3,3-dithio-bis-(sulfosuccinimidyl-propionate), ethylene glycol bis(sulfosuccinimidylsuccinate), dicyclohexyl carbodiimide etc. may be used, but is not limited thereto. On the other hand, when the antigen exhibits activity only when degraded from the polypeptide, a linker that can be cleaved in vivo is used. For example, binders with carboxylic acid esters and/or disulfide bonds may be used.
하나의 구체예에서, 상기 면역세포는 본 발명에 따른 폴리펩타이드에 의해 전달된 항원을 표면에 제시하는 항원제시세포(antigen-presenting cells, APC)일 수 있다. 예를 들어, 상기 면역세포는 수지상세포, 단핵구세포 (monocyte), 랑게르한스(Wrangel Hans) 세포, 마크로파지(macrophage), T세포 또는 B세포일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment, the immune cells may be antigen-presenting cells (APCs) that present antigens delivered by the polypeptide according to the present invention on their surface. For example, the immune cells may be dendritic cells, monocytes, Wrangel Hans cells, macrophages, T cells, or B cells, but are not limited thereto.
상기 면역세포의 표면 분자에 결합하는 펩타이드는 예를 들어, 세포막 투과 펩타이드의 일 말단에 융합될 수 있다. 상기 면역세포의 표면 분자에 결합하는 펩타이드는 세포막 투과 펩타이드의 N-말단 또는 C-말단에 결합될 수 있다. A peptide that binds to the surface molecule of the immune cell may be fused to one end of a cell membrane penetrating peptide, for example. The peptide that binds to the surface molecule of the immune cell may be bound to the N-terminus or C-terminus of the cell membrane penetrating peptide.
경우에 따라서, 상기 면역세포의 표면 분자에 결합하는 펩타이드는 세포막 투과 펩타이드와 링커로 연결될 수 있다. 상기 링커는 펩타이드 링커일 수 있으며, 약 5-25 aa 길이, 구체적으로 약 5-10 aa 길이를 가질 수 있다. 예를 들어, 글리신 및/또는 세린과 같은 친수성 아미노산이 포함될 수 있으나, 이에 제한되는 것은 아니다.In some cases, the peptide binding to the surface molecule of the immune cell may be connected to the cell membrane penetrating peptide through a linker. The linker may be a peptide linker and may have a length of about 5-25 aa, specifically about 5-10 aa. For example, hydrophilic amino acids such as glycine and/or serine may be included, but are not limited thereto.
구체적으로, 상기 링커는 구조적 유연성을 부여하기 위하여 예를 들어, 글리신 링커 (G, Gly)p (p는 1 내지 10), GS 링커 (GnS)m (n, m은 각각 1 내지 10)을 포함할 수 있다. 구체적으로, 상기 링커는 GGGGS 또는 (GGGGS)2를 포함하거나, (G, Gly)p에서 p가 5-10인 5-10 aa의 글리신을 포함할 수 있다. Specifically, the linker is, for example, glycine linker (G, Gly) p (p is 1 to 10), GS linker (G n S) m (n, m is 1 to 10, respectively) to impart structural flexibility. can include Specifically, the linker may include GGGGS or (GGGGS) 2 , or 5-10 aa of glycine where p is 5-10 in (G, Gly)p.
본 발명은 다른 관점에서 상기 폴리펩타이드를 코딩하는 핵산에 관한 것이다. In another aspect, the present invention relates to a nucleic acid encoding the polypeptide.
"핵산"는 DNA(gDNA 및 cDNA) 및 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산에서 기본 구성단위인 뉴클레오타이드는 자연의 뉴클레오타이드 뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다. 본 발명에 따른 핵산의 서열은 변형될 수 있다. 상기 변형은 뉴클레오타이드의 추가, 결실, 또는 비보존적 치환 또는 보존적 치환을 포함한다."Nucleic acid" has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acids, include not only natural nucleotides, but also analogs in which sugar or base sites are modified. . The sequence of a nucleic acid according to the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
상기 핵산을 분리하고, 이를 복제 가능한 벡터 내로 삽입하여 추가로 클로닝하거나 (DNA의 증폭) 또는 추가로 발현시킨다. 이를 바탕으로, 본 발명은 또 다른 관점에서 상기 핵산을 포함하는 벡터에 관한 것이다.The nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or further expression. Based on this, the present invention relates to a vector comprising the nucleic acid in another aspect.
상기 DNA를 통해 통상적인 과정을 사용하여 (예를 들어, DNA와 특이적으로 결합할 수 있는 올리고뉴클레오티드 프로브를 사용함으로써) 결합 단백질의 제1암 및/또는 제2암 코딩 DNA를 용이하게 분리 또는 합성한다. 벡터 성분에는 일반적으로, 다음 중의 하나 이상이 포함되지만, 그에 제한되지 않는다: 신호 서열, 복제 기점, 하나 이상의 마커 유 전자, 증강인자 요소, 프로모터, 및 전사 종결 서열.Through the DNA, the DNA encoding the first arm and/or the second arm of the binding protein is readily isolated using conventional procedures (eg, by using an oligonucleotide probe capable of specifically binding DNA), or synthesize Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
본 명세서에서 사용되는 용어, "벡터"는 숙주세포에서 목적 유전자를 발현시키기 위한 수단으로 플라스미드 벡터; 코즈미드 벡터; 박테리오파지 벡터, 아데노바이러스 벡터, 레트로바이러스 벡터 및 아데노-연관 바이러스벡터 같은 바이러스 벡터 등을 포함한다. 상기 벡터에서 폴리펩타이드를 코딩하는 핵산은 프로모터와 작동적으로 연결되어 있다.As used herein, the term "vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; cosmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. In the vector, the nucleic acid encoding the polypeptide is operably linked to a promoter.
"작동적으로 연결"은 핵산 발현조절서열(예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절하게 된다."Operably linked" means a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence is linked to the other nucleic acid. It will regulate the transcription and/or translation of the sequence.
원핵세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터(예컨대, tac 프로모터, lac 프로모터, lacUV5 프로모터, lpp 프로모터, pLλ 프로모터, pRλ프로모터, rac5 프로모터, amp 프로모터, recA 프로모터, SP6 프로모터, trp 프로모터 및 T7 프로모터 등), 해독의 개시를 위한 라이보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 또한, 예를 들어, 진핵 세포를 숙주로 하는 경우에는, 포유동물 세포의 지놈으로부터 유래된 프로모터(예: 메탈로티오닌 프로모터, β-액틴 프로모터, 사람 헤로글로빈 프로모터 및 사람 근육 크레아틴 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터(예: 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40프로모터, 사이토메갈로바이러스(CMV) 프로모터, HSV의 tk 프로모터, 마우스 유방종양 바이러스(MMTV) 프로모터, HIV의 LTR 프로모터, 몰로니 바이러스의 프로모터엡스타인바 바이러스(EBV)의 프로모터 및 로우스 사코마 바이러스(RSV)의 프로모터)가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 갖는다.When prokaryotic cells are used as hosts, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. Further, for example, when a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (eg, metallotionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or mammalian Promoters derived from animal viruses, such as adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter from HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter from HIV , the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Rouss sarcoma virus (RSV) can be used, and usually has a polyadenylation sequence as a transcription termination sequence.
경우에 따라서, 벡터는 그로부터 발현되는 폴리펩타이드의 정제를 용이하게 하기 위하여 다른 서열과 융합될 수도 있다. 융합되는 서열은, 예컨대 글루타티온 S-트랜스퍼라제(Pharmacia, USA), 말토스 결합 단백질(NEB, USA), FLAG(IBI, USA) 및 6x His(hexahistidine; Quiagen, USA) 등이 있다.In some cases, vectors may be fused with other sequences to facilitate purification of the polypeptides expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).
상기 벡터는 선택표지로서 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함하며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 게네티신, 네오마이신 및 테트라사이클린에 대한 내성 유전자가 있다.The vector contains an antibiotic resistance gene commonly used in the art as a selectable marker, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene.
본 발명은 또 다른 관점에서, 상기 언급된 벡터로 형질전환된 세포에 관한 것이다. 본 발명의 폴리펩타이드를 생성시키기 위해 사용된 세포는 원핵생물, 효모 또는 고등 진핵생물 세포일 수 있으며, 이에 제한되는 것은 아니다. In another aspect, the present invention relates to a cell transformed with the above-mentioned vector. Cells used to produce the polypeptides of the present invention may be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells.
에스케리치아 콜라이(Escherichia coli), 바실러스 서브틸리스 및 바실러스 츄린겐시스와 같은 바실러스 속 균주, 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas)(예를 들면, 슈도모나스 푸티다(Pseudomonas putida)), 프로테우스미라빌리스(Proteus mirabilis) 및 스타필로코쿠스(Staphylococcus)(예를 들면, 스타필로코쿠스 카르노수스(Staphylocus carnosus))와 같은 원핵 숙주세포를 이용할 수 있으나, 이에 제한되는 것은 아니다.Escherichia coli, strains of the genus Bacillus such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (eg, Staphylocus carnosus) may be used, but are not limited thereto.
동물세포의 예로 COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S, 또는 HT1080일 수 있으나, 이에 제한되는 것은 아니다.Examples of animal cells include COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S, or HT1080, but is not limited thereto no.
본 발명은 또 다른 관점에서, 폴리펩타이드에 항원이 결합되어 있는 융합 폴리펩타이드에 관한 것이다.In another aspect, the present invention relates to a fusion polypeptide in which an antigen is linked to the polypeptide.
상기 항원은 암 또는 종양에 대한 면역반응 활성화를 위한 암 또는 종양 항원일 수 있다. 상기 항원은 암 또는 종양 항원으로 세포치료에 있어 암 또는 종양 특이적 면역세포 예를 들어, 세포독성 T 세포를 유도할 수 있다. 상기 암 또는 종양 항원은 예를 들어, 자가(self) 또는 비자가(non-self) 유래 항원일 수 있다. 예를 들면 환자 자신의 유전자에서 유래된 자가 암 또는 종양 항원은 hTERT (GenBank: BAC11010.1), WT1 (GenBank: AAO61088.1), NY-ESO1 (GenBank: CAA05908.1), MAGE-A3 (NCBI Reference Sequence: NP_005353.1) 등과 암 특이적 돌연변이 항원 예를 들면 neoantigens, mutated P53, RAS 등의 종양 억제 또는 유발 유전자를 포함할 수 있으며, 외래 암 또는 종양 항원으로 암 또는 종양 유발 바이러스 항원 예를 들면 CMV, EBV, HPV 등을 포함할 수 있다.The antigen may be a cancer or tumor antigen for activating an immune response against cancer or tumor. The antigen is a cancer or tumor antigen and can induce cancer or tumor specific immune cells, such as cytotoxic T cells, in cell therapy. The cancer or tumor antigen may be, for example, a self or non-self derived antigen. For example, autologous cancer or tumor antigens derived from a patient's own gene include hTERT (GenBank: BAC11010.1), WT1 (GenBank: AAO61088.1), NY-ESO1 (GenBank: CAA05908.1), MAGE-A3 (NCBI Reference Sequence: NP_005353.1) and cancer-specific mutated antigens, such as neoantigens, mutated P53, and RAS, may include tumor suppressor or inducing genes, and cancer or tumor-inducing virus antigens such as foreign cancer or tumor antigens CMV, EBV, HPV, and the like.
자가 암 또는 종양 항원인 hTERT는 염색체 말단에서 텔로미어 DNA(telomeric DNA)를 합성하는 효소로서 암세포는 이 효소를 과도하게 활성화시켜 텔로미어 의존적 세포사멸을 회피할 수 있도록 기능하고 폐암, 위암, 췌장암을 포함하는 다양한 고형암의 타겟 항원으로 알려져 있고 (Kim NW, et al. Science. 1994;266:2011-2015), WT1은 Wilms tumor와 관련된 유전자로서 징크 핑거 전사인자를 암호화하여 세포의 증식과 분화, 자멸사, 기관의 발생에 관여를 하는 단백질로서 뇌척수암, 폐암 등의 타겟 항원으로 알려져 있다 (Call KM, et al., Cell. 1990. 60:509-520; Nakahara Y, et al.,Brain Tumor Pathol. 2004. 21:113-6). 또한 NY-ESO1은 CTA (cancer testis antigen)에 속하는 단백질 중 하나로 주로 생식세포(germ cell)와 육종(sarcoma), 유방암을 포함한 다양한 암세포에 발현하는 것으로 잘 알려져 있다 (Gnjatic S, et al., Adv Cancer Res. 2006;95:1-30). MAGE-A3은 멜라노마 연관 항원 패밀리 (melanoma-associated antigen family)에 속하는 단백질로 폐암, 육종 및 흑색종을 포함한 다양한 암세포에 과발현하는 것으로 알려져 있어 암의 면역치료에 적합한 표적 항원으로 평가되고 있다 (Decoster L, et al.,Ann Oncol. 2012 Jun;23(6):1387-93).hTERT, an autologous cancer or tumor antigen, is an enzyme that synthesizes telomeric DNA at the ends of chromosomes, and cancer cells activate this enzyme excessively to avoid telomere-dependent apoptosis. It is known as a target antigen for various solid cancers (Kim NW, et al. Science. 1994;266:2011-2015), and WT1 is a gene related to Wilms tumor that encodes a zinc finger transcription factor, which causes cell proliferation and differentiation, apoptosis, and organs. As a protein involved in the development of cerebrospinal cancer, it is known as a target antigen for lung cancer, etc. (Call KM, et al., Cell. 1990. 60:509-520; Nakahara Y, et al., Brain Tumor Pathol. 2004. 21:113-6). In addition, NY-ESO1 is one of the proteins belonging to CTA (cancer testis antigen) and is well known to be expressed in various cancer cells, including germ cells, sarcoma, and breast cancer (Gnjatic S, et al., Adv. Cancer Res. 2006;95:1-30). MAGE-A3 is a protein belonging to the melanoma-associated antigen family and is known to be overexpressed in various cancer cells, including lung cancer, sarcoma, and melanoma, and is being evaluated as a suitable target antigen for cancer immunotherapy (Decoster L, et al., Ann Oncol. 2012 Jun;23(6):1387-93).
경우에 따라서, 상기 항원은 예를 들어, A33, ALK, 알파페토단백질(AFP), 아드레날린 수용체 베타 3(ADRB3), alpha-folate receptor, AD034, AKT1, BCMA, 베타-인간 융모막 고나도트로핀, B7H3(CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD79a, CD79b, CD89, CD97, CD123, CD138, CD160, CD171, CD179a, 탄산무수화효소 IX(CAIX), CA-125, 발암배아성 항원(CEA), CCR4, C-유형 렉틴-유사 분자-1(CLL-1 또는 CLECL1), 클라우딘 6(CLDN6), CXORF61, CAGE, CDX2, CLP, CT-7, CT8/HOM-TES-85, cTAGE-1, ERBB2, 상피 성장 인자 수용체(EGFR), EGFR 변이체 III(EGFRvIII), 상피 세포 부착 분자(EPCAM), 신장 인자 2 돌연변이된(ELF2M), 에프린 유형-A 수용체 2(EphA2), EMR2, Fms-유사 티로신 키나제 3(FLT3), FCRL5, Fibulin-1, G250, GD2, 당단백질 36(gp36), 당단백질 100(gp100), 글루코코르티코이드-유도된 종양 괴사 인자 수용체(GITR), GPRC5D, GloboH, G 단백질-결합된 수용체 20(GPR20), GPC3, hsp70-2, 고분자량-흑색종-관련 항원(HMWMAA), A형 간염 바이러스 세포 수용체 1(HAVCR1), HAGE, HCA587/MAGE-C2, hCAP-G, HCE661, HER2/neu, HLA-Cw, HOM-HD-21/Galectin9, HOM-MEEL- 40/SSX2, HOM-RCC-3.1.3/CAXII, HOXA7, HOXB6, Hu, HUB 1, 인슐린 성장 인자(IGF1)-I, IGF-II, IGFI 수용체, 인터류킨-13 수용체 서브유닛 알파-2(IL-13Ra2 또는 CD213A2), 인터류킨 11 수용체 알파(IL-11Ra), IGLL1, KIT(CD117), KM-HN-3, KM-KN- 1, KOC1, KOC2, KOC3, LAGA-1a, LAGE-1, LAIR1, LILRA2, LY75, Lewis Y antigen, MUC1, MN-CA IX, M-CSF, MAGE-1, MAGE-4a, 메소텔린, MAGE-A1, MAD-CT-1, MAD-CT-2, MART1, MPPl 1, MSLN, 신경세포 부착 분자(NCAM), NY-ESO-1, NY-ESO-5, Nkp30, NKG2D, 유선 분화 항원(NY-BR-1), NY-BR-62, NY-BR-85, NY-CO-37, NY-CO-38, NNP-1, NY-LU-12, NY-REN-10, NY-REN-19/LKB/STK1 1, NY-REN-21, NY-REN-26/BCR, NY-REN-3/NY-CO-38, NY-REN-33/SNC6, NY-REN-43, NY-REN-65, NY-REN-9, NY-SAR-35, o-아세틸-GD2 강글리오시드(OAcGD2), OGFr, PSMA, 전립선 산성 포스파타제(PAP), p53, 전립선-암종 종양 항원-1(PCTA-1), 전립선 줄기세포 항원(PSCA), 프로테아제세린 21(테스티신 또는 PRSS21), 혈소판-유래된 성장인자 수용체 베타(PDGFR-beta), PLAC1, 파넥신 3(PANX3), PLU-1, ROR-1, RAGE-1, RU1, RU2, Rab38, RBPJkappa, RHAMM, 단계-특이적 배아 항원-4(SSEA-4), SCP1, SSX3, SSX4, SSX5, Tyrp-1, TAG72, 티로글로불린, 5T4, 종양-관련 당단백질 72(TAG72), 티로시나제(Tyrosinase), 트랜스글루타미나제 5(TGS5), TEM1, TEM7R, 갑상선 자극 호르몬 수용체(TSHR), Tie 2, TRP-2, TOP2A, TOP2B, 유로플라킨 2(UPK2), 비멘틴(Vimentin), 또는 혈관 내피성장 인자 수용체 2(VEGFR2)일 수 있다. Optionally, the antigen is, for example, A33, ALK, alpha-fetoprotein (AFP), adrenergic receptor beta 3 (ADRB3), alpha-folate receptor, AD034, AKT1, BCMA, beta-human chorionic gonadotropin, B7H3 (CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD79a, CD79b, CD89, CD97, CD123, CD138, CD160, CD171 , CD179a, carbonic anhydrase IX (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR4, C-type lectin-like molecule-1 (CLL-1 or CLECL1), claudin 6 (CLDN6), CXORF61 , CAGE, CDX2, CLP, CT-7, CT8/HOM-TES-85, cTAGE-1, ERBB2, epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), epithelial cell adhesion molecule (EPCAM), elongation factor 2 Mutated (ELF2M), Ephrin Type-A Receptor 2 (EphA2), EMR2, Fms-Like Tyrosine Kinase 3 (FLT3), FCRL5, Fibulin-1, G250, GD2, Glycoprotein 36 (gp36), Glycoprotein 100 (gp100), glucocorticoid-induced tumor necrosis factor receptor (GITR), GPRC5D, GloboH, G protein-coupled receptor 20 (GPR20), GPC3, hsp70-2, high molecular weight-melanoma-associated antigen (HMWMAA), Hepatitis A virus cellular receptor 1 (HAVCR1), HAGE, HCA587/MAGE-C2, hCAP-G, HCE661, HER2/neu, HLA-Cw, HOM-HD-21/Galectin9, HOM-MEEL-40/SSX2, HOM -RCC-3.1.3/CAXII, HOXA7, HOXB6, Hu, HUB 1, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2) , interleukin 11 receptor alpha (IL-11Ra), IGLL1, KIT (CD117), KM-HN-3, KM-KN-1, KOC1, KOC2, KOC3, LAGA-1a, LAGE-1, LAIR1, LILRA2, LY75, Lewis Y antigen, MUC1, MN-CA IX, M-CSF, MAGE-1, MAGE-4a, mesothelin, MAGE-A1, MAD-CT-1, MAD-CT-2, MART1, MPPl 1, MSLN, neuron Cell Adhesion Molecule (NCAM), NY-ESO-1, NY-ESO-5, Nkp30, NKG2D, Mammary Differentiation Antigen (NY-BR-1), NY-BR-62, NY-BR-85, NY-CO- 37, NY-CO-38, NNP-1, NY-LU-12, NY-REN-10, NY-REN-19/LKB/STK1 1, NY-REN-21, NY-REN-26/BCR, NY -REN-3/NY-CO-38, NY-REN-33/SNC6, NY-REN-43, NY-REN-65, NY-REN-9, NY-SAR-35, o-acetyl-GD2 ganglio seed (OAcGD2), OGFr, PSMA, prostate acid phosphatase (PAP), p53, prostate-carcinoma tumor antigen-1 (PCTA-1), prostate stem cell antigen (PSCA), protease serine 21 (testicine or PRSS21), platelets -derived growth factor receptor beta (PDGFR-beta), PLAC1, panexin 3 (PANX3), PLU-1, ROR-1, RAGE-1, RU1, RU2, Rab38, RBPJkappa, RHAMM, stage-specific embryonic antigens -4 (SSEA-4), SCP1, SSX3, SSX4, SSX5, Tyrp-1, TAG72, thyroglobulin, 5T4, tumor-associated glycoprotein 72 (TAG72), tyrosinase, transglutaminase 5 (TGS5) ), TEM1, TEM7R, Thyroid Stimulating Hormone Receptor (TSHR), Tie 2, TRP-2, TOP2A, TOP2B, Europlakin 2 (UPK2), Vimentin, or Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) can
또한, 상기 항원은 병원균 (박테리아, 바이러스 등), 화합물질, 꽃가루, 암세포, 새우 등 또는 이들의 일부 펩타이드 또는 단백질이며, 더욱 바람직하게는 암 항원 펩타이드이나, 체내에서 면역 반응을 일으킬 수 있는 물질이라면 이에 제한되지 않는다. 상기 항원은 바람직하게는 단백질, 재조합 단백질, 당단백질, 유전자, 펩티드, 다당류, 지질다당류, 폴리뉴클레오티드, 세포, 세포 용해물(lysate), 박테리아, 바이러스 등일 수 있으며, 더욱 바람직하게는, 암 항원 펩타이드일 수 있다. 상기 당백질은 항체, 항체의 단편, 구조 단백질, 조절 단백질, 전사인자, 독소 단백질, 호르몬, 호르몬 유사체, 효소, 효소의 단편, 수송 단백질, 수용체(receptor), 수용체의 단편, 생체방어 유도 물질, 저장 단백질, 이동 단백질(movement protein), 익스플로이티브 단백질(exploitive protein), 리포터 단백질 등일 수 있다. 그러나 생체에서 항원으로 작용하여 면역 반응을 유도할 수 있는 물질이라면 이에 제한되지 않는다.In addition, the antigen is a pathogen (bacterium, virus, etc.), a chemical substance, pollen, cancer cell, shrimp, etc., or some peptide or protein thereof, more preferably a cancer antigen peptide, or a substance that can cause an immune response in the body Not limited to this. The antigen may preferably be a protein, recombinant protein, glycoprotein, gene, peptide, polysaccharide, lipopolysaccharide, polynucleotide, cell, cell lysate, bacterium, virus, etc., more preferably, a cancer antigen peptide can be The protein includes antibodies, antibody fragments, structural proteins, regulatory proteins, transcription factors, toxin proteins, hormones, hormone analogues, enzymes, enzyme fragments, transport proteins, receptors, fragments of receptors, biological defense inducers, It may be a storage protein, a movement protein, an exploitive protein, a reporter protein, and the like. However, any material capable of inducing an immune response by acting as an antigen in a living body is not limited thereto.
상기 폴리펩타이드와 항원은 공유적 또는 비공유적 결합을 통해 연결되어, 복합체를 형성하고, 면역세포에 포함되도록 함으로써 항원으로 면역세포를 감작시킬 수 있다. The polypeptide and the antigen are linked through a covalent or non-covalent bond, form a complex, and are included in the immune cell, thereby sensitizing the immune cell with the antigen.
본 발명에 따른 상기 융합 폴리펩타이드를 코딩하는 핵산에 관한 것이다. 상기 폴리펩타이드에 항원이 결합되어 있는 융합 폴리펩타이드는 벡터를 통해 전달될 수 있다. It relates to nucleic acids encoding the fusion polypeptides according to the present invention. A fusion polypeptide in which an antigen is bound to the polypeptide may be delivered through a vector.
본 발명은 또한 상기 융합 폴리펩타이드 및 상기 핵산을 포함하는 면역세포에 관한 것이다. 상기 융합 폴리펩타이드의 항원을 통해 면역세포가 감작되도록 할 수 있다. The present invention also relates to an immune cell comprising said fusion polypeptide and said nucleic acid. Immune cells can be sensitized through the antigen of the fusion polypeptide.
상기 감작은 항원이 면역세포에 전달되어 세포 표면상에 로딩되도록 하는 것을 의미한다. 상기 항원은 폴리펩타이드와 재조합 항원 형태로 제조되어, 면역세포에 감작될 수 있다. 폴리펩타이드 및 상기 폴리펩타이드에 결합된 항원을 면역세포에 24시간 이하 동안 처리함으로써, 면역세포를 감작시킬 수 있다. 감작시간은 항원의 종류 및 면역세포의 종류에 따라 조절될 수 있다. The sensitization means that the antigen is delivered to immune cells and loaded onto the cell surface. The antigen may be prepared in the form of a polypeptide and a recombinant antigen, and sensitize immune cells. Immune cells can be sensitized by treating immune cells with the polypeptide and the antigen bound to the polypeptide for 24 hours or less. The sensitization time may be adjusted according to the type of antigen and the type of immune cell.
상기 항원은 본 발명에 따른 폴리펩타이드와 결합할 수 있는 항원으로, 불활성화 종양세포, 유전자 재조합 방법에 의해 제조된 종양세포 관련 유전자, 펩타이드 또는 단백질을 포함할 수 있다. 유전자 재조합 방법에 의해 상기 항원을 수득하고자 하는 경우, 상기 항원을 인코딩하는 뉴클레오타이드 서열은 공지일 수 있으며, 공지된 서열의 전장을 이용할 수 있으나, 전장의 일부를 이용할 수도 있다. 상기 항원을 인코딩하는 뉴클레오타이드 서열을 벡터에 클로닝하여 목적하는 항원이 발현되도록 할 수 있다.The antigen is an antigen capable of binding to the polypeptide according to the present invention, and may include inactivated tumor cells, tumor cell-related genes, peptides or proteins produced by genetic recombination methods. When obtaining the antigen by genetic recombination, the nucleotide sequence encoding the antigen may be known, and the full length of the known sequence may be used, but a part of the full length may also be used. A nucleotide sequence encoding the antigen may be cloned into a vector to express the desired antigen.
상기 항원은 폴리펩타이드의 N-말단 또는 C-말단에 결합할 수 있다. 공유결합 방법은 항원의 종류에 따라 당업계에 공지된 방법을 이용하여 실시할 수 있으며, 예를 들어 폴리펩타이드를 인코딩하는 유전자를 클로닝 및 세포 내 발현시켜 수득할 수 있다.The antigen may bind to the N-terminus or C-terminus of the polypeptide. The covalent bonding method may be performed using a method known in the art depending on the type of antigen, and may be obtained, for example, by cloning and intracellularly expressing a gene encoding a polypeptide.
경우에 따라서, 폴리펩타이드의 활성을 방해하지 않는 링커, 예를 들어 N-숙시니미딜 요오도아세테이드, N-말레이미도부티릴 옥시숙신아미드 에스테르, 1,5-디플루오로-2,4-디니트로벤젠, 디스디아조벤지딘, 3,3-디티오-비스-(설포숙시니미딜-프로피오네이트), 에틸렌 글리콜비스(설포숙시니미딜숙시네이트), 디시클로헥실 카보디이미드 등이 이용될 수 있으나, 이에 한정되지 아니한다. 한편, 항원이 폴리펩타이드로부터 분해되었을 때에만 활성을 나타내는 경우에는, 링커는 생체 내에서 절단 가능한 것을 사용한다. 예를 들어, 카복실산 에스테르 및/또는 디설파이드 결합이 있는 결합제가 이용될 수 있다.Optionally, a linker that does not interfere with the activity of the polypeptide, for example N-succinimidyl iodoacetate, N-maleimidobutyryl oxysuccinamide ester, 1,5-difluoro-2,4 -Dinitrobenzene, disdiazobenzidine, 3,3-dithio-bis-(sulfosuccinimidyl-propionate), ethylene glycol bis(sulfosuccinimidylsuccinate), dicyclohexyl carbodiimide etc. may be used, but is not limited thereto. On the other hand, when the antigen exhibits activity only when degraded from the polypeptide, a linker that can be cleaved in vivo is used. For example, binders with carboxylic acid esters and/or disulfide bonds may be used.
하나의 구체예에서, 상기 면역세포는 본 발명에 따른 폴리펩타이드에 의해 전달된 항원을 표면에 제시하는 항원제시세포(antigen-presenting cells, APC)일 수 있다. 예를 들어, 상기 면역세포는 수지상세포, 단핵구세포 (monocyte), 랑게르한스(Wrangel Hans) 세포, 마크로파지(macrophage), T세포 또는 B세포일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment, the immune cells may be antigen-presenting cells (APCs) that present antigens delivered by the polypeptide according to the present invention on their surface. For example, the immune cells may be dendritic cells, monocytes, Wrangel Hans cells, macrophages, T cells, or B cells, but are not limited thereto.
본 발명은 또 다른 관점에서, 상기 면역세포를 포함하는 면역치료제에 관한 것이다. 본 발명에 따른 면역치료제는 면역 반응을 증가시키거나, 특정 질병, 감염 또는 질환의 치료 또는 예방에 바람직한 면역 반응의 일부를 선택적으로 상승시킬 수 있다.In another aspect, the present invention relates to an immunotherapeutic agent comprising the immune cells. An immunotherapeutic agent according to the present invention may increase an immune response or selectively increase a portion of an immune response desirable for treatment or prevention of a particular disease, infection or disorder.
이에 기반하여, 본 발명은 또 다른 관점에서, 상기 면역세포를 포함하는 항-종양 또는 항암 백신에 관한 것이다. 본 발명에 따른 백신을 통해 객체의 면역원성을 증가시킬 수 있으므로, 이를 통해 객체 내 종양의 증식 및/또는 전이를 예방 또는 억제할 수 있다.Based on this, in another aspect, the present invention relates to an anti-tumor or anti-cancer vaccine comprising the immune cells. Since the immunogenicity of a subject can be increased through the vaccine according to the present invention, proliferation and/or metastasis of a tumor in the subject can be prevented or suppressed.
상기 백신은 일회 투여함으로써 수행되는 면역화 방법과 연속 투여함으로써 수행되는 면역화 방법을 모두 포함할 수 있다.The vaccine may include both an immunization method performed by one-time administration and an immunization method performed by continuous administration.
본 발명은 또 다른 관점에서, 종양 또는 암 치료용 조성물에 관한 것이다. In another aspect, the present invention relates to a composition for treating tumors or cancer.
본 발명의 조성물에 포함되는 유효성분 등의 함량 및 투여방법은 통상의 환자의 증후와 질병의 심각도에 기초하여 본 기술분야의 통상의 전문가가 결정할 수 있다. 단위-투여량 또는 다-투여량 용기, 예를 들면 밀봉된 앰플 및 병 등으로 제공될 수도 있다.The amount and administration method of the active ingredient included in the composition of the present invention can be determined by a person skilled in the art based on the symptoms and severity of the disease of a typical patient. It may also be presented in unit-dose or multi-dose containers, such as sealed ampoules and bottles.
본 발명의 조성물은 경구 또는 비경구 투여가 가능하다. 본 발명에 따른 조성물의 투여경로는 이들로 한정되는 것은 아니지만, 예를 들면, 기관지, 구강, 정맥 내, 근육 내, 동맥 내, 골수 내, 경막 내, 심장 내, 경피, 피하, 복강 내, 장관, 설하 또는 국소 투여가 가능하다. 본 발명에 따른 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 방법, 배설율 또는 질병의 중증도 등에 따라 그 범위가 다양하며, 본 기술분야의 통상의 전문가가 용이하게 결정할 수 있다. 또한, 임상 투여를 위해 공지의 기술을 이용하여 본 발명의 조성물을 적합한 제형으로 제제화할 수 있다.The composition of the present invention can be administered orally or parenterally. The administration route of the composition according to the present invention is not limited to these, but for example, bronchial, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal , sublingual or topical administration is possible. The dosage of the composition according to the present invention varies in its range depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate or severity of disease, etc. can decide In addition, the composition of the present invention can be formulated into a suitable dosage form for clinical administration using known techniques.
본 발명의 조성물은 약제학적 조성물로, 약제학적으로 허용되는 담체를 포함할 수 있다. 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.The composition of the present invention is a pharmaceutical composition and may include a pharmaceutically acceptable carrier. As those commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 투여 시간 및 투여 경로와 같은 요인들에 의해 다양하게 처방될 수 있으나 투여량에 따른 심각한 독성(Grade 3 이상)은 보고되지 않았기 때문에 제조의 방법 및 수율에 따라서 많은 부분이 결정된다. 한편, 본 발명의 약제학적 조성물의 피내 또는 피하 투여량은 바람직하게는 1회당 0.1X107~10X107 세포이다.A suitable dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, administration time and administration route, but severe toxicity (Grade 3 or more) has not been reported, so much is determined by the manufacturing method and yield. Meanwhile, the intradermal or subcutaneous dosage of the pharmaceutical composition of the present invention is preferably 0.1X107 to 10X107 cells per dose.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조된다. 이때 제형은 세포 동결용 용액에 현탁 또는 완충용액에 현탁하는 형태일 수도 있으며, 안정화제를 추가적으로 포함할 수 있다. 본 발명의 일 실시예에 따른 면역세포는 항원 감작 이후 동결시키고, 필요시 해동하여 사용할 수 있다. The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using pharmaceutically acceptable excipients according to a method that can be easily performed by those skilled in the art. At this time, the formulation may be in the form of suspension in a cell freezing solution or suspension in a buffer solution, and may additionally include a stabilizer. Immune cells according to an embodiment of the present invention may be frozen after antigen sensitization and thawed if necessary.
활성 성분의 투여량은 투여 경로, 환자의 연령, 성별, 체중 및 환자의 중증도 등의 여러 인자에 따라 적절히 선택될 수 있고, 상기 조성물은 종양 또는 암 증상을 예방, 개선 또는 치료하는 효과를 가지는 공지의 화합물과 병행하여 투여할 수 있다.The dosage of the active ingredient may be appropriately selected according to various factors such as the route of administration, age, sex, weight and severity of the patient, and the composition is known to have an effect of preventing, improving or treating tumors or cancer symptoms. It can be administered in parallel with the compound of
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 펩타이드를 이용한 결합력 시험 연구Example 1. Binding force test study using peptides
1.1 펩타이드 합성1.1 Peptide Synthesis
MHC 클래스 Ⅱ와 결합하는 슈퍼 항원 펩타이드 (superantigen peptide) 중 5종을 선별하여 펩타이드 제작 업체인 애니잰㈜에 합성 의뢰를 진행하여 펩타이드 5종을 합성하였다.Five kinds of superantigen peptides that bind to MHC class II were selected and synthesized by requesting synthesis from Anisan Co., Ltd., a peptide manufacturer, and five kinds of peptides were synthesized.
1.1.1 ELISA 활용 시험1.1.1 ELISA Utilization Test
합성된 펩타이드의 결합력을 확인하기 위해 96 웰 플레이트에 MHC 클래스 ±단백질을 코팅 한 후 펩타이드를 50nM로 처리 한 다음 24시간 인큐베이션을 진행하였다. 이후 3회 PBS 워싱 후 HRP-항체(1:500)를 2시간 인큐베이션 진행한 뒤 5회 PBS 워싱 이후 TMB 용액을 넣은 후 10~30min 인큐베이션 후 정지 용액 (stopping solution: 2M H2SO4)활용 발색을 정지시킨 후 흡광도 450nm에서 확인하여 결합력이 좋은 펩타이드를 선별하였다. 그 결과 합성한 펩타이드 5가지중 SEB2에 해당하는 펩타이드 서열이 가장 높은 결합력을 보였으며 그 다음으로 SEB4, SEB1, SEB3 순서로 결합력을 보였다. In order to confirm the binding force of the synthesized peptide, a 96-well plate was coated with MHC class ± protein, and then the peptide was treated with 50 nM and incubated for 24 hours. Then, after 3 times PBS washing, HRP-antibody (1:500) was incubated for 2 hours, after 5 times PBS washing, TMB solution was added, and after 10~30min incubation, color development using a stopping solution (2M H 2 SO 4 ) After stopping, the absorbance was confirmed at 450 nm to select peptides with good binding ability. As a result, among the five synthesized peptides, the peptide sequence corresponding to SEB2 showed the highest binding ability, followed by SEB4, SEB1, and SEB3 in order.
1.1.2 PBMC를 이용 단핵구 유래 수지상세포 (monocyte-derived dendritic cell) 분화 1.1.2 Differentiation of monocyte-derived dendritic cells using PBMC
PBMC 바이얼을 꺼내서 해동 시킨 후 1%AB RPMI 배지를 활용하여 T150 플레이트에 30분동안 단핵구를 부착시켰다. 이후 부착되지 않은 세포들은 제거 한 후 성숙화 배지인 hGM-CSF 30ng/ml, hIL-4 20ng/ml로 3일간 배양 진행하였다.After the PBMC vial was taken out and thawed, monocytes were adhered to a T150 plate for 30 minutes using 1% AB RPMI medium. Thereafter, non-adherent cells were removed and cultured for 3 days in the maturation medium hGM-CSF 30ng/ml and hIL-4 20ng/ml.
1.1.3 펩타이드 세포투과성 측정1.1.3 Measurement of peptide cell permeability
MoDC 세포는 30-40 % 컨플루언트(confluent) 상태로 커버슬립(cover slip)에 분주한 뒤, 1 x 104 cells/well의 세포 밀도로 6 웰 플레이트에 보관되었다. 펩타이드를 각각 농도에 따라 처리 후 배양한 뒤 PBS로 세척하고 상온에서 15분 동안 4%파라포름알테하이드로 고정하였다. 이후 3% BSA를 이용하여 차단 (blocking) 진행 후 세포 내에 투과된 펩타이드를 표시하기 위해 FITC, DAPI를 이용하여 염색을 진행, 이후 형광현미경을 이용 분석 진행하였다. 그 결과 기존 원천특허로써 가지고 있는 CTP 서열 펩타이드 대비 APC의 결합력이 높은 SEB2를 결합한 CTP-SEB2 서열 펩타이드가 moDC세포에서 월등히 많이 투과되는 것을 관찰 할 수 있었으며, 그해 반에 SEB2 서열 펩타이드만 처리한 군에서는 투과되는 현상을 관찰할 수 없었다. 위 결과를 도 1에 나타내었다. MoDC cells were seeded on coverslips in a 30-40% confluent state and stored in 6-well plates at a cell density of 1 x 10 4 cells/well. The peptides were treated and incubated according to each concentration, washed with PBS, and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Thereafter, after blocking using 3% BSA, staining was performed using FITC and DAPI to display peptides penetrated into cells, and then analysis was performed using a fluorescence microscope. As a result, it was observed that the CTP-SEB2 sequence peptide, which combines SEB2 with high APC binding ability, compared to the CTP sequence peptide possessed as the original original patent, permeated much more in moDC cells. Permeation could not be observed. The above results are shown in Figure 1.
1.1.4 유동세포분석기를 이용한 세포투과성 측정1.1.4 Measurement of cell permeability using flow cytometry
MoDC는 인간말초혈액에서 단핵구를 분리 후 배양하여 수지상 세포를 수득하였다. 분화된 수지상세포를 이용하여 각 그룹별로 펩타이드 농도에 따라 처리 후 10분간 배양하였으며, 이후 PBS를 이용 각각의 그룹을 세척하였다. 수지상세포의 표현형을 분석하기 위해 항-마우스 CD80, 항-마우스 CD86, 항-마우스 CD11c, 펩타이드-FITC를 각각 염색한 후 FACS CantoⅡ를 이용하여 측정하였다. 먼저 제조한 MoDC의 표현형을 확인 후 제조한 DC가 DC로써의 기능을 하는지 확인하는 분석을 진행 하였으며 그 결과 모든 DC 에서 90%이상의 표현형을 나타내었음을 확인하였다. 그 후 DC안에서의 펩타이드 투과성을 분석하기 위해 CD11c+와 CD80+의 이중 집단 (double population)을 수득한 후 그 안에서의 펩타이드-FITC를 분석하였다. 분석 결과 현미경 결과와 동일하게 CTP-SEB2 펩타이드 그룹에서 기존 CTP 펩타이드 그룹대비 많은 양의 펩타이드가 투과된 것을 확인할 수 있었으며, SEB2 단독 펩타이드의 경우는 투과성이 거의 없는 것을 확인하였다. 그 결과를 도 2에 나타내었다. MoDC was obtained by culturing dendritic cells after isolating monocytes from human peripheral blood. Using differentiated dendritic cells, each group was treated according to the peptide concentration and cultured for 10 minutes, and then each group was washed with PBS. To analyze the phenotype of dendritic cells, anti-mouse CD80, anti-mouse CD86, anti-mouse CD11c, and peptide-FITC were stained, respectively, and then measured using FACS Canto II. First, after confirming the phenotype of the prepared MoDC, an analysis was performed to confirm that the prepared DC functioned as a DC, and as a result, it was confirmed that all DCs showed a phenotype of 90% or more. Then, in order to analyze peptide permeability in DC, a double population of CD11c+ and CD80+ was obtained and peptide-FITC therein was analyzed. As a result of the analysis, it was confirmed that a large amount of peptides permeated in the CTP-SEB2 peptide group compared to the existing CTP peptide group in the same way as the microscopic result, and it was confirmed that there was almost no permeability in the case of SEB2 alone peptide. The results are shown in FIG. 2 .
실시예 2. CTP가 융합된 펩타이드 융합단백질 제작Example 2. Preparation of peptide fusion protein in which CTP is fused
2.1 융합단백질 벡터 제작.2.1 Construction of fusion protein vectors.
투과성 결과에서 결합력이 가장 우수한 SEB2 서열 펩타이드를 선정하여 유전자재조합 단백질 벡터를 도 3과 같이 제작 의뢰(genscript, Piscataway, NJ)하였다.A recombinant protein vector was commissioned (genscript, Piscataway, NJ) to produce a recombinant protein vector as shown in FIG.
2.2 재조합 융합단백질 생산.2.2 Production of recombinant fusion proteins.
재조합된 유전자를 E.coli를 이용하여 배양을 진행 한 뒤 IB상태로 제작한 이후 가용화 단계를 거쳐 Ni-NTA 레진 (resin)을 이용하여 분리 정제를 진행하였다. 구체적으로는 먼저 37℃에서 하룻밤 동안 종균 배양한 후, LB Broth Miller 배지 (Novagen, Yeast extract 5g,peptone from casein 10g, sodium chloride 10g)에 앰피실린(Ampicillin) 50μg/ml 을 첨가한 배양액 속에서 37℃로 키웠다. OD600 nm의 흡광도에서 그 값이 0.8~1.0 정도에 도달했을 때, 최종 0.5mM의 농도가 되도록 IPTG를 넣어 단백질의 발현을 유도시킨 후 180rpm에서 8시간 동안 진탕 배양하였다. OD600nm의 흡광도에서 그 값이 1.5~2.0 정도로 자란 박테리아 세포들은 10,000rpm, 4℃에서 20분간 원심분리하여 침전시킨 후, 침전된 세포2g (1L culture volume)당 50ml의 붕해 완충액(300mM NaCl, 50mM Tris-HCl (pH 8.0), 0.5% Triton X-100)으로 세포가 완전히 현탁될 때까지 교반시켰다. PMSF를 최종 농도 1mM이 되게 넣은 후, 초음파 분쇄기로 세포를 융해하였다. 융해된 세포는 12000rpm, 4℃에서 30분간 원심분리 하여 현탁액과 총 융해물을 분리한 후, 현탁액만을 모아 0.45μm의 기공 크기의 셀룰로오스 여과 막에 여과하여 미리 평형 완충액으로 평형화시킨 니켈 친화성 크로마토그래피 컬럼에 로딩하였다. 정제된 단백질을 버퍼 교환을 통해 안정화 상태로 만든 뒤 QC를 진행하였다. 그 결과를 도 4에 나타내었다. The recombinant gene was cultured using E.coli, produced in an IB state, and then separated and purified using Ni-NTA resin through a solubilization step. Specifically, after culturing the seed at 37 ° C overnight, 37 grown at °C. When the absorbance at OD600 nm reached about 0.8 to 1.0, protein expression was induced by adding IPTG to a final concentration of 0.5 mM, followed by incubation with shaking at 180 rpm for 8 hours. Bacterial cells grown to a value of 1.5 to 2.0 at an absorbance of OD600 nm were precipitated by centrifugation at 10,000 rpm and 4 ° C for 20 minutes, and then 50 ml of disintegration buffer (300 mM NaCl, 50 mM Tris) per 2 g (1 L culture volume) of the precipitated cells. -HCl (pH 8.0), 0.5% Triton X-100) and the cells were agitated until completely suspended. After adding PMSF to a final concentration of 1 mM, the cells were lysed with an ultrasonicator. The lysed cells were centrifuged at 12000 rpm, 4℃ for 30 minutes to separate the suspension and the total lysate, and then collected only the suspension and filtered through a cellulose filtration membrane with a pore size of 0.45 μm, and equilibrated with an equilibration buffer beforehand. Nickel affinity chromatography loaded onto the column. After the purified protein was stabilized through buffer exchange, QC was performed. The results are shown in FIG. 4 .
2.3 융합단백질 세포투과성 측정2.3 Measurement of fusion protein cell permeability
MoDC 세포는 30-40 % 컨플루언트(confluent) 상태로 커버슬립(cover slip)에 분주한 뒤, 1 x 104 cells/well의 세포 밀도로 6 well plate에 보관되었다. 펩타이드를 각각 농도에 따라 처리 후 배양한 뒤 PBS로 세척하고 상온에서 15분 동안 4%파라포름알테하이드로 고정하였다. 이후 3% BSA를 이용하여 blocking진행 후 세포 내에 투과된 단백질을 표시하기 위해 Cy5.5, DAPI를 이용하여 염색을 진행, 이후 형광현미경을 이용 분석 진행. 재조합 단백질 미처리군 대비 처리군인 CTP-SEB2-OVA 그룹에서 많은 양의 단백질이 투과된 것을 확인 할 수 있었으며, 기존 CTP 서열로 재조된 CTP-OVA 단백질 처리 그룹보다도 투과량이 많은 것을 확인 할 수 있었다. 그에 반해 CTP가 포함되지 않은 OVA, SEB-OVA 그룹의 경우 단백질 투과가 안 되는 것을 확인 할 수 있었다. 그 결과를 도 5에 나타내었다. MoDC cells were seeded onto coverslips at 30-40% confluent and stored in 6 well plates at a cell density of 1 x 104 cells/well. The peptides were treated and incubated according to each concentration, washed with PBS, and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Subsequently, after blocking using 3% BSA, staining was performed using Cy5.5 and DAPI to display proteins permeated into cells, and then analysis was performed using a fluorescence microscope. It was confirmed that a large amount of protein permeated in the CTP-SEB2-OVA group, which is a treatment group compared to the recombinant protein untreated group, and the permeation amount was higher than that of the CTP-OVA protein treated group prepared with the existing CTP sequence. On the other hand, in the case of OVA and SEB-OVA groups without CTP, it was confirmed that protein permeation was not possible. The results are shown in FIG. 5 .
2.4 웨스턴 블롯 활용 융합단백질 세포투과성 측정2.4 Measurement of fusion protein cell permeability using Western blot
세포를 차가운 PBS로 세척한 후, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM DTT, 30 mM NaF, 10 mM Na3VO4,0.5% NP40 및 Pierce (protease inhibitor cocktail)를 포함하는 용배버퍼 (lysis buffer)에 의해 용해시켰다. 세포 전체 용해물 (Whole cell lysates)은 Bradford 제제 (Bio-Rad)를 이용하여 동일한 농도로 조절하였으며, 40-120 μg의 용해물을 사용하여 8-12% 아크릴아마이드 겔 (acrylamide gel)에 SDS-PAGE를 진행하였으며 PVDF 멤브레인 (Milipore)로 이동하였다. 면역블롯팅 (immunoblotting)을 진행하기 위해, 멤브레인을 0.5% Tween20이 포함된 TBS (TBST)로 제조된 5% non-fat dry milk로 상온에서 한 시간 동안 차단 (blocking) 하였다. 멤브레인을 TBST로 4회 세척하고 4% non-fat dry milk 로 희석한 1차 항체를 최적의 농도로 처리한 후 4℃에서 오버나이트 (overnight)하여 배양하였다. TBST로 4회 세척 후, HRP가 결합된 2차 항체와 함께 45분 동안 배양하였다. 결합된 항체는 chemiluminescent HRP substrate (Millipore, USA) 및 Chemi DOC (Bio-RAD, USA)를 이용하여 검출하였다After washing the cells with cold PBS, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM DTT, 30 mM NaF, 10 mM Na3VO4, 0.5% NP40 and Pierce (protease inhibitor cocktail) buffer containing (lysis buffer). Whole cell lysates were adjusted to the same concentration using Bradford preparation (Bio-Rad), and 40-120 μg of lysate was used to 8-12% acrylamide gel (SDS- It was subjected to PAGE and transferred to a PVDF membrane (Milipore). To proceed with immunoblotting, the membrane was blocked with 5% non-fat dry milk prepared in TBS (TBST) containing 0.5% Tween20 at room temperature for one hour. The membrane was washed 4 times with TBST, treated with an optimal concentration of primary antibody diluted in 4% non-fat dry milk, and incubated overnight at 4°C. After washing four times with TBST, the cells were incubated with HRP-conjugated secondary antibodies for 45 minutes. Bound antibodies were detected using chemiluminescent HRP substrate (Millipore, USA) and Chemi DOC (Bio-RAD, USA).
2.5 융합단백질의 직접적인 결합력 분석2.5 Direct binding force analysis of fusion proteins
선별된 펩타이드를 활용 HIS-tag이 발현되는 유전자를 제작하여 단백질을 제조하여 MHC 클래스 Ⅱ와 직접적인 상호작용을 하는지의 여부를 알아보기 위하여, His에 대한 면역침강법(immunoprecipitation)을 실시하여 분석하였다.In order to determine whether a gene expressing an HIS-tag using the selected peptide was prepared to produce a protein and directly interact with MHC class II, His was analyzed by immunoprecipitation.
구체적으로, 4X10^7개의 세포를 수거하여 PBS로 2회 세척한 후, 단백질 분해효소 억제제 칵테일[2 mM AEBSF, 1mM EDTA, 130 μM 베스타틴(Bestatin), 1 μM 레우펩틴(leupeptin), 14 μM E-64, 0.3 μM 아프로티닌(Aprotinin)]과 탈인산가수분해효소 억제제(Roche)를 포함한 NP-40 용해 완충용액(lysis buffer)(20 mM Tris,pH 7.4, 150 mM NaCl, 1% NP-40, 10% 글리세롤)로 부유한 세포를, 4℃ 챔버에서 회전자(rotator)에 끼워 30분간 돌려주었다(C1, 25 rpm). 13000 rpm, 4℃에 10분 동안 원심분리하고, 상층액을 수거한 다음, 다시 12000rpm, 4℃에 10분 동안 원심분리하여 깨끗한 상층액만을 수거하였다. 단백질을 정량한 뒤, 세포파쇄물 데이타(cell lysate data)로 사용할 단백질은 따로 샘플링(sampling)하여 -80℃에 보관 해놓고, 면역침강에 사용할 단백질은 500 ㎍ 이상/700 ~ 1000 ㎕으로 덜고 총 1 ml이 되도록 나머지는 용해 완충용액으로 채웠다. 여기에 면역침강용 항체를 단백질 100 ~ 500 ㎍ 당 1 ~ 2 ㎍씩 넣고 4℃ 챔버에서 회전자에 끼워 2시간 이상 또는 밤새 돌려주었다(C1, 10 rpm). 그런 다음 단백질 A/G 비드(Protein A/G beads)(Pierce)를 약 30 ~ 50 ㎕ 넣고 1 ~ 2시간 4℃ 챔버에서 회전자로 돌려주었다. NP-40 용해 완충용액으로 1200 rpm, 3min, 4℃씩 3회에 걸쳐 세척하였다. 세척 후에는 잔여 완충액을 완전히 제거하고 5X샘플 완충용액(sample buffer)을 20 ㎕ 넣고 끓였다. 얼음에서 식힌 후 원심분리기로 2분간 돌린 후, 상층액만 따서 7.5 ~ 12% SDSPAGE 법으로 분리하였다. 겔(gel)은 다시 PVDF 멤브레인으로 블럿하고, 단백질이 옮겨진 멤브레인에 1차 항체를 붙인 후 2차 항체 IgG에 대한 항체를 붙인 후 ECL 탐색 키트(detection kit)로 관찰하였다.Specifically, after harvesting 4X10^7 cells and washing them twice with PBS, protease inhibitor cocktail [2 mM AEBSF, 1 mM EDTA, 130 μM Bestatin, 1 μM leupeptin, 14 μM E-64, 0.3 µM Aprotinin] and NP-40 lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40) containing dephosphatase inhibitors (Roche). 40, 10% glycerol), the cells were put on a rotator in a 4 ° C chamber and rotated for 30 minutes (C1, 25 rpm). Centrifuged at 13000 rpm, 4°C for 10 minutes, collected the supernatant, and then centrifuged again at 12000 rpm, 4°C for 10 minutes to collect only the clear supernatant. After quantifying the protein, the protein to be used as cell lysate data is sampled separately and stored at -80 ° C. The remainder was filled with lysis buffer solution. Here, 1 to 2 μg of antibody for immunoprecipitation was added per 100 to 500 μg of protein, put on a rotator in a 4° C. chamber, and rotated for at least 2 hours or overnight (C1, 10 rpm). Then, about 30 to 50 μl of Protein A/G beads (Pierce) was added and returned to the rotor in a chamber at 4° C. for 1 to 2 hours. NP-40 lysis buffer solution was washed three times at 1200 rpm, 3 min, and 4 ° C. After washing, the remaining buffer was completely removed, and 20 μl of 5X sample buffer was added and boiled. After cooling on ice and centrifuging for 2 minutes, only the supernatant was collected and separated by 7.5-12% SDSPAGE method. The gel was again blotted with a PVDF membrane, and after attaching a primary antibody to the protein-transferred membrane, an antibody to the secondary antibody IgG was attached, and then observed with an ECL detection kit.
분석결과를 도 6에 나타내었다. 그 결과, protein A/G 비드 (bead)와 융합단백질을 결합시켜 타켓 단백질로 웨스턴 블랏 (western blot)을 진행 하였을 경우 펩타이드가 융합된 단백질에서 타겟 단백질과 함께 검출되는 것을 확인할 수 있으며, 펩타이드가 없는 단백질에서는 검출되지 않는 것을 확인할 수 있었다.The analysis results are shown in FIG. 6 . As a result, when Western blot was performed with the target protein by combining the protein A/G beads with the fusion protein, it was confirmed that the peptide was detected together with the target protein in the fused protein. It was confirmed that the protein was not detected.
2.6 마우스 수지상 세포 분리 2.6 Isolation of mouse dendritic cells
수지상 세포(DC)는 골수줄기세포 (bone marrow stem cells)를 수지상세포로 분화시켜 배양하였다. 보다 구체적으로, 골수 유래 수지상 세포 (Bone marrow-derived DC, BMDC)는 6-8주령 female C57BL/6 마우스의 대퇴골과 경골로부터 골수 세포를 분리한 후, 적혈구를 제거하기 위해 ACK lysis buffer (Lonza)를 처리하였다. 세포를 세척하고 10 ng/ml mGM-CSF (Creagene Inc.)를 포함하는 세포 배양배지 (10% FBS와 penicillin/streptomycin이 포함된 RPMI 1640)로 배양하였다. 2일 후, 배양된 세포의 상층액을 제거한 후 mGM-CSF가 포함된 새로운 배양배지 2 ml로 교체하였다. 배양 4일째, mGM-CSF가 포함된 새로운 배양배지 1 ml을 첨가하였다. 배양 6일째, 비접착성 세포들을 수거하여 미성숙 수지상세포 (immature DCs, imDC)로 사용하였다. 성숙 수지상 세포(mature DC, mDC)는 미성숙 수지상 세포(immature DC)를 LPS (100~200ng/ml) 존재 하에 24 시간동안 배양하여 준비하였다Dendritic cells (DC) were cultured by differentiating bone marrow stem cells into dendritic cells. More specifically, bone marrow-derived DC (BMDC) was isolated from the femur and tibia of 6-8 week old female C57BL/6 mice, and then incubated with ACK lysis buffer (Lonza) to remove red blood cells. was processed. The cells were washed and cultured in a cell culture medium (RPMI 1640 containing 10% FBS and penicillin/streptomycin) containing 10 ng/ml mGM-CSF (Creagene Inc.). After 2 days, the supernatant of the cultured cells was removed and replaced with 2 ml of a new culture medium containing mGM-CSF. On the 4th day of culture, 1 ml of fresh culture medium containing mGM-CSF was added. On day 6 of culture, non-adherent cells were harvested and used as immature DCs (imDC). Mature dendritic cells (mature DC, mDC) were prepared by culturing immature dendritic cells (immature DC) in the presence of LPS (100-200ng/ml) for 24 hours.
2.7 유세포분석기를 이용한 세포투과성 측정2.7 Measurement of cell permeability using flow cytometry
BMDC는 마우스 뼈에서 단핵구를 분리 후 배양하여 수지상 세포를 수득하였다. 분화된 수지상세포를 이용하여 각 그룹별로 펩타이드 농도에 따라 처리 후 1시간 배양하였으며, 이후 PBS를 이용 각각의 그룹을 세척하였다. 수지상세포의 표현형을 분석하기 위해 항-마우스 CD80, anti-mouse CD86, 항-마우스 CD11c를 각각 염색한 후 FACS CantoⅡ를 이용하여 측정하였으며, CD11c+중 펩타이드 투과성을 확인하기위해 펩타이드-FITC로 분석을 진행하였다.BMDC was cultured after isolating monocytes from mouse bones to obtain dendritic cells. Using differentiated dendritic cells, each group was cultured for 1 hour after treatment according to the peptide concentration, and then each group was washed with PBS. In order to analyze the phenotype of dendritic cells, anti-mouse CD80, anti-mouse CD86, and anti-mouse CD11c were stained and measured using FACS CantoII, and analysis was performed with peptide-FITC to confirm peptide permeability in CD11c+. did
2.8 TCA침전법을 이용한 배양액내 단백질 잔존량 분석2.8 Analysis of remaining protein in the culture medium using the TCA precipitation method
배양액 시료에 100% TCA용액을 1/10볼륨으로 첨가한 후 강하게 볼텍싱(vortexing) 후 얼음속에서 30분간 방치 후 4℃, 14,000 rpm에서 10분간 원심분리하였다. 이후 상등액을 버리고 침전물에 콜드 아세톤 (cold acetone) 500ul 첨가하여 4℃, 14,000 rpm에서 10분간 원심분리 후 TCA를 제거하였다. 이후 침전물을 말린 후 완충용액을 사용하여 재현탁 후 웨스턴 블롯 진행하였다. 도 7에서와 같이, 배지상에 존재하는 단백질 잔존량을 확인 한결과 펩타이드가 결합된 CTP-SEB2-OVA 단백질의 경우 대부분 세포내로 침투하여 배지에 존재하는 단백질 양이 많지 않은 반면, CTP-OVA 기존 서열 단백질의 경우 투과량이 조금 낮은 것을 확인 할 수 있었다. 그러나, CTP가 결합되지 않은 단백질들의 경우 모두 배지상에 존재하는 것을 확인할 수 있었다. A 100% TCA solution was added to the culture sample at a volume of 1/10, followed by strong vortexing, and then left in ice for 30 minutes, followed by centrifugation at 4°C and 14,000 rpm for 10 minutes. Thereafter, the supernatant was discarded, and 500 ul of cold acetone was added to the precipitate, followed by centrifugation at 4° C. and 14,000 rpm for 10 minutes, and then TCA was removed. Then, the precipitate was dried, resuspended using a buffer solution, and Western blotting was performed. As shown in FIG. 7, as a result of confirming the amount of protein remaining on the medium, most of the peptide-conjugated CTP-SEB2-OVA protein penetrated into the cells and the amount of protein present in the medium was not large, whereas the CTP-OVA existing In the case of the sequence protein, it was confirmed that the permeation amount was slightly low. However, it was confirmed that all of the proteins to which CTP was not bound were present on the medium.
2.9 PTD계열 펩타이드 세포막 투과성 비교 실험2.9 PTD series peptide cell membrane permeability comparison experiment
기존에 알려진 PTD계열의 투과성 펩타이드 서열에 현재 자사가 개발하고자 하는 펩타이드 서열을 접목하였을 때의 투과성 비교를 통해 자사가 개발하는 서열이 좀더 투과성이 우월하다는 것을 확인하고자 하였다. BMDC 세포를 12-웰 플레이트에 2x105/ml의 갯수로 시딩 (seeding)한 후, 1일간 37℃에서 배양하였다. 세포 표면에 부착된 1uM의 농도로 CTP-FITC, TAT-SEB2-FITC 및 CTP-SEB2-FITC 펩타이드 (애니젠㈜)를 4시간동안 세포에 처리하고 세포의 형광표지 정도를 FACS로 조사하였다. 우선 마우스 BMDC에서의 DC 표현형들을 분석한 결과 모두 90% 이상의 수율로써 BMDC가 잘 제조가 된 것을 확인할 수 있으며, 해당 DC에 펩타이드를 처리한 결과 기존에 알려진 PTD 계열 펩타이드에 SEB2 서열을 접목하여 투과된 양보다 자사가 보유하고 있는 CTP서열에 SEB2를 접목하였을 때 그 효과가 더 크다는 것을 확인 할 수 있었다. 이는 융합단백질 결과에서도 동일하게 확인되었다 (도 8).Through the comparison of permeability when the peptide sequence to be developed by the company was grafted to the previously known penetrating peptide sequence of the PTD series, it was confirmed that the sequence to be developed by the company has superior permeability. BMDC cells were seeded in a 12-well plate at a number of 2×10 5 /ml, and then cultured at 37° C. for 1 day. Cells were treated with CTP-FITC, TAT-SEB2-FITC and CTP-SEB2-FITC peptides (Anigen Co., Ltd.) at a concentration of 1 uM attached to the cell surface for 4 hours, and the degree of fluorescence labeling of the cells was examined by FACS. First, as a result of analyzing DC phenotypes in mouse BMDC, it was confirmed that BMDC was well prepared with a yield of 90% or more. It was confirmed that the effect was greater when SEB2 was grafted onto the CTP sequence owned by the company than the amount. This was also confirmed in the fusion protein results (FIG. 8).
2.10 항원 특이적 T cell 증식 분석2.10 Antigen-specific T cell proliferation assay
OVA (ovalbumin) 항원에 특이적인 TCR(OT-I) 트랜스제닉 마우스 (transgenic mice)로써 해당 마우스 비장을 이용하여 DC와 공배양을 통한 T 세포 증식 반응 분석하였다. 배양 세포의 기능 확인을 위한 MLR 측정은 96-웰 플랫 밑바닥에 마이크로타이터 장착된 플레이트 (96-well flat-bottomed microtiter plate)에 비장세포(R, OT-1 specific T cell,: 1X105 cells/well)와 OVA 항원으로 처리된 DCs (S, stimulator)의 비율(R:S ratio)을 1:10∼1:100로 하여 96시간 함께 배양한 후 반응기 (responder) 세포의 증식 정도를 측정하였다. 도 9에서와 같이, 펩타이드가 융합된 항원로 감작된 DC가 들어간 그룹에서 OVA 특이적 T 세포 반응이 가장 높았으며 CTP 항원 감작 DC의 경우는 특이적 T 세포 반응이 증가하기는 하였지만 펩타이드 융합 항원보다는 반응성이 낮은 것을 확인하였다. As OVA (ovalbumin) antigen-specific TCR (OT-I) transgenic mice (transgenic mice), the T cell proliferation response was analyzed through co-culture with DC using the mouse spleen. For MLR measurement to confirm the function of cultured cells, splenocytes (R, OT-1 specific T cell,: 1X105 cells/well) were placed in a 96-well flat-bottomed microtiter plate. ) and OVA antigen-treated DCs (S, stimulator) at a ratio (R:S ratio) of 1:10 to 1:100 and incubated together for 96 hours, and then the degree of responder cell proliferation was measured. As shown in FIG. 9, the OVA-specific T cell response was the highest in the group containing DC sensitized with the peptide-fused antigen, and although the specific T-cell response increased in the case of CTP antigen-sensitized DC, it was higher than that of the peptide fusion antigen. It was confirmed that the reactivity was low.
실시예 3. 수지상세포 백신에 의한 마우스 흑색종 예방효과Example 3. Mouse melanoma preventive effect by dendritic cell vaccine
수지상세포 백신에 의한 마우스 흑색종 예방효과(Prevention model) 수지상세포 백신에 의한 흑색종 예방 가능성을 조사하기 위하여, CTP-융합 흑색종 재조합 항원을 감작시킨 수지상세 포를 마우스에 2차례 면역시키고 그 후에 흑색종 특이 항원을 발현하는 암세포주로 도전시험을 실시하여 고형암 형성 여부를 조사하였다. 마우스 수지상세포는 대퇴골과 경골의 골수세포를 수지상세포로 분화시켜 사용하였다. 대퇴골과 경골의 양끝을 절단하고 골수세포를 추출하여 50 ㎖ 튜브에 세포를 수거하였다. 수거된 골수세포를 0.83% 염화암모늄 용액으로 현탁시켜 적혈구를 제거하고 골수세포를 수지상세포 생산배지(RPMI-1640,10% FBS, 10 ng/㎖의 마우스 재조합 IL-4와 10 ng/㎖의 마우스 GM-CSF)에서 2일간 배양하며 비흡착성 세포를 제거하고 용기 바닥에 부착된 세포만을 취하였다. 2-3일 간격으로 신 선한 배지로 교체하여 사이토카인의 고갈을 방지하였다. 배양 6일째 미성숙 수지상세포를 수거하고 여기에 재조합 항원인 CTP-OVA와 SCTP-OVA를 각각 처리하였다. 각 항원 단백질을 10 ㎍/㎖ 한국등록특허 제10-0647847호)씩 20시간 처리하여 미성숙 수지상세포를 감작하였으며 24시간부터 수지상세포 성숙화에 필요한 사이토카인(100 ㎍/㎖ IFN-γ와 100 ㎍/㎖ TNF-α)을 첨가하여 수지상세포의 성숙화를 유도하였다. 항원으로 감작된 수지상세포 1x 106 세포를 마우스 피하로 주사하여 항암면역을 유도하였다. 수지상세포 면역은 1주 간격으로 2회 접종하였으며, 2차 수지상세포 접종 1주 후에 B16-OVA melanoma cell를 각 1x 106 세포/마우스로 SC (subcutaneous) 주사하였다. 암의 크기(가로 x 세로)는 매 2일마다 측정하였다. 측정결과, CTP-OVA 또는 SCTP-OVA 항원으로 감작된 수지상세포를 접종시킨 실험군에서는 도 10에서와 같이 항종양 효과를 확인할 수 있었다.Mouse melanoma prevention effect by dendritic cell vaccine (Prevention model) To investigate the possibility of melanoma prevention by dendritic cell vaccine, mice were immunized twice with dendritic cells sensitized with CTP-fusion melanoma recombinant antigen, and then A challenge test was conducted with a cancer cell line expressing a melanoma-specific antigen to examine whether solid cancer was formed. Mouse dendritic cells were used by differentiating bone marrow cells of the femur and tibia into dendritic cells. Both ends of the femur and tibia were cut, bone marrow cells were extracted, and the cells were collected in a 50 ml tube. The collected bone marrow cells were suspended in 0.83% ammonium chloride solution to remove red blood cells, and the bone marrow cells were dendritic cell production medium (RPMI-1640, 10% FBS, 10 ng/ml mouse recombinant IL-4 and 10 ng/ml mouse GM-CSF) for 2 days, and non-adherent cells were removed, and only cells attached to the bottom of the container were taken. Depletion of cytokines was prevented by replacing the culture medium with a fresh medium every 2-3 days. On day 6 of culture, immature dendritic cells were collected and treated with the recombinant antigens CTP-OVA and SCTP-OVA, respectively. Immature dendritic cells were sensitized by treating each antigenic protein at 10 μg/ml Korean Patent No. 10-0647847) for 20 hours, and cytokines necessary for dendritic cell maturation (100 μg/ml IFN-γ and 100 μg/ml IFN-γ) were sensitized from 24 hours. ml TNF-α) was added to induce maturation of dendritic cells. Anticancer immunity was induced by subcutaneously injecting dendritic cells 1x10 6 cells sensitized with the antigen into mice. Dendritic cell immunization was inoculated twice at 1-week intervals, and 1 week after the second dendritic cell inoculation, B16-OVA melanoma cells were SC (subcutaneous) injected into each 1x10 6 cell/mouse. Cancer size (width x height) was measured every 2 days. As a result of the measurement, the antitumor effect was confirmed as shown in FIG. 10 in the experimental group inoculated with dendritic cells sensitized with CTP-OVA or SCTP-OVA antigen.
실시예 4. PBMC를 이용 면역세포 투과성 분석Example 4. Analysis of immune cell permeability using PBMC
PBMC상에 존재하는 여러 면역세포(monocyte, T cell, B cell, Dendritic cell, NK cell등)들에서 개발하고자 하는 융합펩타이드의 투과성을 관찰하고자 하였다. 그러기 위해 동결된 PBMC를 해동 후 기존 CTP 펩타이드와 융합된 펩타이드 CTP-SEB펩타이드를 각각 1uM 농도로 처리 후 FACS로 분석을 진행하였다. 각각의 면역세포들의 경우 면역세포들의 대표적인 마커인 CD14(monocyte), CD3(T cell), CD19(B cell), HLA-DR(Dendritic cell)등으로 확인 후 투과된 펩타이드 양을 분석하였다. 면역세포별 펩타이드 투과성을 확인한 결과, 도 11에서와 같이 HLA-DR을 발현한다고 알려진 단핵구세포 (monocyte), B 세포 (B cell), 수지상세포 (dendritic cell)에서 기존 CTP 펩타이드 대비 융합 펩타이드가 투과성이 증가되는 것을 확인할 수 있었으며, 추가적으로 T세포 (T cell)에서도 투과량이 증가되는 것을 확인할 수 있었다. 이를 통해 전반적으로 융합 펩타이드가 면역세포에서 투과량이 증가되는 것을 확인할 수 있었다. The permeability of the fusion peptide to be developed was observed in various immune cells (monocyte, T cell, B cell, Dendritic cell, NK cell, etc.) existing on PBMC. To do so, the frozen PBMC was thawed, and the CTP-SEB peptide fused with the existing CTP peptide was treated at a concentration of 1 uM, respectively, and analyzed by FACS. In the case of each immune cell, CD14 (monocyte), CD3 (T cell), CD19 (B cell), HLA-DR (dendritic cell), etc., which are representative markers of immune cells, were identified and the amount of permeated peptide was analyzed. As a result of confirming the peptide permeability of each immune cell, as shown in FIG. 11, the fusion peptide is permeable compared to the existing CTP peptide in monocyte, B cell, and dendritic cell known to express HLA-DR It was confirmed that the permeation amount was increased, and it was confirmed that the amount of permeation was additionally increased in T cells. Through this, it was confirmed that the permeation amount of the fusion peptide was increased in the immune cells as a whole.
본 발명에 따른 폴리펩타이드는 우수한 면역세포의 세포막 투과 능력을 가지며, 면역세포에 직접적인 타켓팅을 통해 우수한 효율로 항원이 전달되도록 함으로써, T 세포 증식에 효과적일 수 있다. The polypeptide according to the present invention has an excellent ability to penetrate the cell membrane of immune cells, and can be effective in T cell proliferation by allowing antigens to be delivered with excellent efficiency through direct targeting to immune cells.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
전자파일 첨부하였음.Electronic file attached.
Claims (21)
- 세포막 투과 펩타이드 (cytoplasmic transduction peptide, CTP) 및 면역세포의 표면 분자에 결합하는 펩타이드를 포함하는, 면역세포에 항원을 전달하기 위한 폴리펩타이드. A polypeptide for delivering an antigen to immune cells, including a cytoplasmic transduction peptide (CTP) and a peptide that binds to surface molecules of immune cells.
- 제1항에 있어서, 상기 면역세포의 표면 분자는 MHC (major histocompatibility complex) 클래스 II (MHC class II)인 폴리펩타이드.The polypeptide according to claim 1, wherein the surface molecule of the immune cell is a major histocompatibility complex (MHC) class II.
- 제1항에 있어서, 상기 면역세포는 수지상세포, 단핵구세포 (monocyte), 랑게르한스(Wrangel Hans) 세포, 마크로파지(macrophage), T세포 또는 B세포인 폴리펩타이드.The polypeptide according to claim 1, wherein the immune cells are dendritic cells, monocytes, Wrangel Hans cells, macrophages, T cells or B cells.
- 제1항에 있어서, 상기 면역세포의 표면 분자에 결합하는 펩타이드는 서열번호 1 내지 4로 구성된 군에서 선택되는 서열을 포함하는 폴리펩타이드.The polypeptide according to claim 1, wherein the peptide binding to surface molecules of immune cells comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 4.
- 제1항에 있어서, 상기 세포막 투과 펩타이드는 서열번호 5 내지 7로 구성된 군에서 선택되는 서열을 포함하는 폴리펩타이드.The polypeptide according to claim 1, wherein the cell membrane penetrating peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 5 to 7.
- 제1항에 있어서, 상기 세포막 투과 펩타이드의 말단에 면역세포의 표면 분자에 결합하는 펩타이드가 융합된, 폴리펩타이드.The polypeptide according to claim 1, wherein a peptide binding to surface molecules of immune cells is fused to the terminal of the cell membrane penetrating peptide.
- 제1항에 있어서, 상기 항원은 암 또는 종양에 대한 면역반응 활성화를 위한 암 또는 종양 항원인 폴리펩타이드.The polypeptide according to claim 1, wherein the antigen is a cancer or tumor antigen for activating an immune response against cancer or tumor.
- 제1항 내지 제7항 중 어느 한 항에 따른 폴리펩타이드를 코딩하는 핵산.A nucleic acid encoding a polypeptide according to any one of claims 1 to 7.
- 제1항 내지 제7항 중 어느 한 항에 따른 폴리펩타이드에 항원이 결합되어 있는 융합 폴리펩타이드. A fusion polypeptide wherein an antigen is bound to the polypeptide according to any one of claims 1 to 7.
- 제9항에 있어서, 상기 항원은 암 또는 종양에 대한 면역반응 활성화를 위한 암 또는 종양 항원인 융합 폴리펩타이드.The fusion polypeptide according to claim 9, wherein the antigen is a cancer or tumor antigen for activating an immune response against cancer or tumor.
- 제9항의 융합 폴리펩타이드를 코딩하는 핵산.A nucleic acid encoding the fusion polypeptide of claim 9.
- 제9항의 융합 폴리펩타이드를 포함하는 면역세포.An immune cell comprising the fusion polypeptide of claim 9.
- 제12항에 있어서, 수지상세포, 단핵구세포 (monocyte), 랑게르한스(Wrangel Hans) 세포, 마크로파지(macrophage), T세포 또는 B세포인 면역세포.The immune cell according to claim 12, which is a dendritic cell, a monocyte, a Wrangel Hans cell, a macrophage, a T cell or a B cell.
- 제12항의 면역세포를 포함하는 면역치료제.An immunotherapeutic agent comprising the immune cells of claim 12.
- 제12항의 면역세포를 포함하는 항-종양 또는 항암 백신.An anti-tumor or anti-cancer vaccine comprising the immune cells of claim 12.
- 제12항의 면역세포를 포함하는 종양 또는 암 치료용 조성물.A composition for treating tumor or cancer comprising the immune cells of claim 12.
- 제11항의 핵산을 포함하는 면역세포.An immune cell comprising the nucleic acid of claim 11.
- 제17항에 있어서, 수지상세포, 단핵구세포 (monocyte), 랑게르한스(Wrangel Hans) 세포, 마크로파지(macrophage), T세포 또는 B세포인 면역세포.The immune cell according to claim 17, which is a dendritic cell, a monocyte, a Wrangel Hans cell, a macrophage, a T cell or a B cell.
- 제17항의 면역세포를 포함하는 면역치료제.An immunotherapeutic agent comprising the immune cells of claim 17.
- 제17항의 면역세포를 포함하는 항-종양 또는 항암 백신.An anti-tumor or anti-cancer vaccine comprising the immune cells of claim 17.
- 제17항의 면역세포를 포함하는 종양 또는 암 치료용 조성물.A composition for treating tumor or cancer comprising the immune cells of claim 17.
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